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Rice protein improves oxidative stress by regulating glutathione metabolism and attenuating oxidative damage to lipids and proteins in rats
Life Sciences 91 (2012) 389–394
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Rice protein improves oxidative stress by regulating glutathione metabolism and attenuating oxidative damage to lipids and proteins in rats Lin Yang a,⁎, Jia-Hou Chen b, Tong Xu c, Ai-Shen Zhou b, Hong-Kun Yang b a b c
School of Food Science and Engineering, Harbin Institute of Technology, 73 Huanghe Road, Harbin 150090, China Heilongjiang Provincial Environmental Monitoring Central Station, Harbin 150056, China Heilongjiang Provincial Hospital, Harbin 150036, China
a r t i c l e
i n f o
Article history: Received 3 January 2012 Accepted 2 August 2012 Keywords: Rice protein Oxidative Antioxidative Glutathione Lipid Rats
a b s t r a c t Aims: To evaluate the effects of rice protein (RP) on glutathione metabolism and oxidative damage. Main methods: Seven-week-old male Wistar rats were fed diets containing casein and RP without cholesterol for 3 weeks. Plasma and liver lipid levels, hepatic accumulation of total glutathione (T-GSH), oxidized glutathione (GSSG), reduced glutathione (GSH), malondialdehyde (MDA) and protein carbonyl (PCO) were measured. In the liver, the total antioxidative capacity (T-AOC), mRNA levels of glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modulatory subunit (GCLM), and the activities of hepatic catalase (CAT), total superoxide dismutase (T-SOD), γ-glutamylcysteine synthetase (γ-GCS), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GSHPx) were also measured. Key findings: T-AOC, GCLC and GCLM mRNA levels, antioxidative enzyme activities (T-SOD and CAT) and glutathione metabolism related enzyme activities (γ-GCS, GST, GR and GSHPx) were effectively stimulated by RP feeding compared to casein, and RP significantly reduced the hepatic accumulation of MDA and PCO in rats. These results indicate that lipid-lowering activity was induced by RP feeding. Significance: The present study demonstrates that RP improves oxidative stress primarily through enzymatic and non-enzymatic antioxidative defense mechanisms, reflected by enhancing the antioxidative status and attenuating the oxidative damage to lipids and proteins. These results suggest that RP can prevent hyperlipidemia in part through modifying glutathione metabolism, and sulfur amino acids may be the main modulator of this antioxidative mechanism. © 2012 Elsevier Inc. All rights reserved.
Introduction Oxidative stress is one of the major risk factors for developing hyperlipidemia. Diet plays an important role in regulating oxidative stress to prevent hyperlipidemia (Avula and Fernandes, 1999; Coyle et al., 2008; Gorinstein et al., 2005; Lee, 2006). Accordingly, the suppression of oxidative stress may be a useful target for new therapies in preventing hyperlipidemia. Oxidative stress can be caused by oxidative damage to lipids and proteins. In addition to malondialdehyde (MDA), which is generally used as a marker of lipid peroxidation, the oxidation process may be accelerated by the formation and accumulation of carbonylated protein (Pirinccioglu et al., 2010). Recent studies have shown that protein carbonylation may be involved in various disease states, and protein carbonyls (PCO) may serve as biomarkers of oxidative stress (Dalle-Donne et al., 2003). Thus, lipid peroxidation and protein ⁎ Corresponding author at: Department of Food Science, School of Food Science and Engineering, Harbin Institute of Technology, 73 Huanghe Road, Nangang District, Harbin 150090, China. Tel.: +86 451 8628 2910; fax: +86 451 8628 2906. E-mail address: [email protected] (L. Yang). 0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2012.08.003
oxidative damage may indicate a higher risk for developing hyperlipidemia, suggesting that effective dietary antioxidants should be not only reactive oxygen species scavengers but also reactive carbonyl scavengers. Glutathione (GSH) is an antioxidant that is ubiquitous in mammals, and it plays important roles in several detoxification reactions and in the suppression of lipid peroxidation (Anderson, 1998). The liver represents the major site of GSH metabolism, in which GSH and its related enzymes comprise an antioxidant defense system that protects against oxidative stress. Further, as a non-essential nutrient, GSH can be synthesized in the body from several amino acids (L-cysteine, L-glutamic acid and glycine), suggesting that the amino acid profile of the diet may be a major contributor to GSH metabolism responsible for an antioxidative defense. Rice is a staple cereal that is widely consumed around the world (Kishine et al., 2008; Ohtsubo and Nakamura, 2007; Tran et al., 2005). There has been a growing emphasis on the improvement of the physiological functions of rice (Tran et al., 2004; Yang et al., 2012a), and an association between rice protein consumption and reduced plasma and liver lipid levels has been extensively demonstrated in some studies (Yang et al., 2007, 2012b; Yang and Kadowaki, 2009). However, the
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precise mechanism by which rice protein affects lipid metabolism has not been fully established. Specifically, there is not yet a comprehensive understanding of the link between the modulation of oxidative status and the consumption of rice protein. Accordingly, evidence describing how rice protein can improve oxidative stress to regulate lipid metabolism is lacking. The present study, therefore, was conducted to focus on the regulatory effects of rice protein on GSH metabolism and lipid and protein oxidative damage status. The key questions addressed are: (1) whether there is a link between lipid-lowering action and antioxidative stress and the consumption of rice protein and (2) whether rice protein can improve oxidative stress in growing rats fed cholesterol-free diets.
At the end of the feeding period, the rats were deprived of food for 18 h and then sacrificed. Blood was withdrawn from the abdominal vein into a heparinized syringe under anesthesia with sodium pentobarbital (50 mg/kg body weight), immediately cooled on ice and separated by centrifugation at 12,000 ×g for 5 min. The plasma obtained was frozen at − 20 °C until analysis. After blood collection, the liver was excised immediately, rinsed in saline and weighed after blotting on filter paper. The whole liver was cut into several portions and quickly freeze-clamped in liquid nitrogen and stored at − 80 °C until analysis.
Materials and methods
Plasma free fatty acid (FFA), triglyceride (TG), total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) concentrations were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Plasma non-high-density lipoprotein cholesterol (non-HDL-C) was calculated as non-HDL-C = TC − HDL-C. The lipids in the liver were extracted and purified as described by Folch et al. (1957) and analyzed as described by Yang et al. (2011a). Samples of liver were extracted with chloroform/methanol (2:1, v/v). Hepatic total cholesterol, triglyceride and free fatty acid levels were measured with a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Protein sources Rice protein (RP) from Oryza sativa L. cv. Longjing 26 (Rice Research Institute of Heilongjiang Academy of Agricultural Sciences, Jiamusi, China) and casein (CAS) (Gansu Hualing Industrial Group, Gansu, China) were used as the dietary protein sources in the present study. RP was prepared by the alkaline extraction method (Yang et al., 2011a, 2012b). Animals and diets The present experiment was performed in compliance with the Guidelines of the Committee for Animal Experimentation of Harbin Medical University and followed the same protocol used in previous studies (Yang et al., 2011a, 2012b). Seven-week-old male Wistar rats were purchased from the Animal Center of Harbin Medical University (Harbin, China) and individually housed in metabolic cages in a room maintained at 22 ± 2 °C under a 12 h light–dark cycle (07:00–19:00 light). Rats were allowed free access to commercial pellets (Animal Center of Harbin Medical University, Harbin, China) for 3 days. After acclimatization, the rats were randomly divided into two groups of similar body weight. Each group consisted of six rats. All animals were fed ad libitum with experimental diets according to the formula recommended by the American Institute of Nutrition (Reeves et al., 1993). For 3 weeks, growing rats were fed cholesterolfree diets with a dietary protein level of 20% (as crude protein, CP) of CAS and RP, respectively. Diets were completed to 100% with starch. The compositions of the experimental diets are shown in Table 1.
Analyses of plasma and liver lipid levels
Determination of malondialdehyde and protein carbonyl The hepatic malondialdehyde (MDA) and protein carbonyl (PCO) contents were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Estimation of hepatic total antioxidative capacity The hepatic total antioxidative capacity (T-AOC) was measured with a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Measurements of reduced glutathione and oxidized glutathione Total glutathione (T-GSH) and oxidized glutathione (GSSG) in the liver were assayed with commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Reduced glutathione (GSH) was calculated as GSH = T-GSH − 2 × GSSG.
Sample collection Analyses of hepatic enzyme activities During the feeding period, food consumption and body weight were recorded daily in the morning before replenishing the diets.
Table 1 Composition of the experimental diets (g/kg diet).
CAS RP Sucrose Cellulose Soybean oil β-Cornstarch Mineral mixc Vitamin mixd Choline bitartrate Tert-butylhydroquinone L-Cysteine a b c d
CASa
RPb
228.9 – 100.0 50.0 70.0 500.6 35.0 10.0 2.5 0.014 3.0
– 221.8 100.0 50.0 70.0 510.7 35.0 10.0 2.5 0.014 –
CAS, casein, crude protein 873.7 g/kg. RP, rice protein, crude protein 901.7 g/kg. Mineral mixture, AIN-93G-MX (Nosan Corp., Japan). Vitamin mixture, AIN-93-VX (Nosan Corp., Japan).
The activities of hepatic total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSHPx), glutathione reductase (GR), glutathione-S-transferase (GST) and γ-glutamyl cysteine synthetase (γ-GCS) were determined using kits from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Quantitative real-time PCR Total RNA was extracted from rat livers using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. cDNA was reverse transcribed from 1 μg of total RNA using a PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Bio. Inc., Otsu, Shiga, Japan). For quantitative real time PCR, cDNA was analyzed with the ABI 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) using SYBR Green (Takara Bio. Inc., Otsu, Shiga, Japan). The primer sequences used were: 5′-ACAGCAACAGGGTGGT GG-AC-3′ (forward) and 5′-TTTGAGGGTGCAGCGAACTT-3′ (reverse) for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH); 5′-CC TCCTCCTCCAAACTCAGAT-A-3′ (forward) and 5′-CCACAAATACCACAT
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AGGCAGA-3′ (reverse) for rat glutamate cysteine ligase catalytic subunit (GCLC); 5′-GGGCACAGGTAAAACCCAATA-3′ (forward) and 5′-TTCAATGTCAGGGATGCTTTC-3′ (reverse) for rat glutamate cysteine ligase modulatory subunit (GCLM). The results were normalized to the level of GAPDH mRNA. Statistical analysis The data are presented as the means ± SEM. Differences between groups were examined for statistical significance using one-way analysis of variance (ANOVA) and then determined with the least significant difference test. The criterion for significance was P b 0.05.
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different from that in the CAS group (P > 0.05). Like the TG concentration, the plasma FFA level was also 14.47% lower in the RP group than in the CAS group (P > 0.05). The hepatic total cholesterol concentrations were significantly lower in rats fed RP than those fed CAS (P b 0.05) (Table 2), consistent with previous studies (Yang et al., 2007, 2011a). In parallel, hepatic triglyceride levels decreased by 35.22% in RP compared to CAS (P b 0.05). As shown in Table 2, the significant reduction in hepatic triglyceride deposits was mainly reflected by the diminished concentrations of free fatty acids in the RP group. However, no significant difference in free fatty acid concentration was found between the CAS and RP groups (P > 0.05), although RP feeding reduced hepatic free fatty acid levels by 13.02%.
Results Malondialdehyde and protein carbonyls Body weight, food intake and liver weight Body weight gain was significantly reduced in rats fed RP compared with CAS (P b 0.05) (Table 2). No significant change in food intake was found between RP and CAS, suggesting that the dietary protein did not affect food intake during the growing period. As shown in Table 2, less liver swelling was observed in rats fed RP (P b 0.05) than in those fed CAS, as suggested by previous studies (Yang et al., 2007, 2011a).
Total antioxidative capacity
CAS
RP
21.33 ± 0.63 5.28 ± 0.24 8.85 ± 0.33
19.98 ± 0.35 4.27 ± 0.26⁎ 7.83 ± 0.26⁎
1.51 ± 0.03 0.93 ± 0.06 0.58 ± 0.05 0.62 ± 0.09 0.41 ± 0.03 0.44 ± 0.03 0.76 ± 0.03
1.34 ± 0.02⁎ 0.95 ± 0.05 0.39 ± 0.04⁎ 0.41 ± 0.06⁎
15.07 ± 0.50 58.49 ± 2.21 29.73 ± 1.36
11.00 ± 0.44⁎ 37.89 ± 1.96⁎ 25.86 ± 1.14
0.35 ± 0.02 0.37 ± 0.03 0.65 ± 0.03
Values within the same row with ⁎ are significantly different (P b 0.05). a Values are means ± SEM for six rats per group.
5 4
*
3 2 1 0 CAS
B Protein carbonyls contents (nmol/mg protein)
Table 2 Food intake, body weight gain, liver weight, plasma and liver lipids of rats fed experimental diets.a
A
C
RP
4
*
3 2 1 0 CAS
T-AOC (units/min per mg protein)
As shown in Table 2, RP significantly reduced the plasma TC concentration, by 11.26%, compared with CAS (P b 0.05), showing that RP exhibited an effective hypocholesterolemic activity in growing rats. Concomitant with the decrease in plasma TC, the plasma non-HDL-C concentration was also distinctly lower in rats fed RP than in those fed CAS (P b 0.05), whereas the HDL-C level did not differ significantly between experimental groups (P > 0.05). As a result, the ratio of non-HDL-C/HDL-C was significantly decreased, by 33.87%, in the RP group compared with the CAS group (P b 0.05). The results indicated that RP-feeding effectively altered the cholesterol distribution, reflected mainly by the lower concentration of non-HDL-C in the RP group. Accompanying the decreased level of TC, the plasma TG concentration was also reduced by RP feeding in this study, compared with CAS (Table 2). However, no significant difference in TG level was observed between the CAS and RP groups, despite the fact that RP feeding lowered the TG by 14.63% (P > 0.05). The TG to HDL ratio also decreased in the RP group (− 15.91%) but was not significantly
MDA contents (nmol/mg protein)
As shown in Fig. 1C, hepatic total antioxidative capacity (T-AOC) was significantly stimulated by RP in growing rats fed cholesterol-free diets
Plasma lipids, lipoprotein profiles and hepatic lipids
Food intake (g/day) Body weight gain (g/day) Liver weight (g) Plasma lipids (mmol/L) Cholesterol TC HDL-C Non-HDL-C Non-HDL-C/HDL-C (mol/mol) Triglyceride TG/HDL-C (mol/mol) Free fatty acids Hepatic lipids (μmol/g liver) Cholesterol Triglyceride Free fatty acids
After 3 weeks of feeding, the hepatic accumulations of MDA (Fig. 1A) and PCO (Fig. 1B) were significantly altered by the dietary protein treatments, showing a marked difference in the oxidative damage to lipid and protein between CAS and RP. The MDA and PCO contents were significantly decreased when rats were fed an RP diet (MDA: − 23.02%; PCO: − 21.34%) (P b 0.05).
2
RP
*
1.6 1.2 0.8 0.4 0 CAS
RP
Fig. 1. The effects of dietary proteins on the hepatic contents of lipid peroxidation products (A), protein carbonyls (B) and hepatic total antioxidative capacity (C) in 7-week-old male Wistar rats fed cholesterol-free diets for 3 weeks. The values shown represent the means ± SEM (n = 6). Bars marked with ⁎ are significantly different (P b 0.05). CAS, casein; MDA, malondialdehyde; RP, rice protein; T-AOC, total antioxidative capacity.
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(P b 0.05). Compared with CAS, RP feeding produced a marked increase in T-AOC (27.54%), suggesting that RP feeding could stimulate the antioxidative status under the present experimental conditions.
Glutathione contents As illustrated in Fig. 2, hepatic glutathione contents were effectively regulated by dietary proteins. Compared with CAS, the hepatic concentration of GSH (Fig. 2A) was increased 40.91% by RP feeding (P b 0.05), whereas the accumulation of GSSG in the liver was depressed by 18.06% in the RP group (Fig. 2B) (P b 0.05). As a result, a pronounced reduction in the ratio of GSSG to GSH was produced by RP compared to CAS (Fig. 2C) (P b 0.05).
Hepatic enzyme activities As summarized in Table 3, the activities of various hepatic enzymes involved in antioxidative activities and glutathione metabolism were affected by the dietary protein. Compared with CAS, T-SOD was enhanced 60.07% by RP feeding (P b 0.05). Similarly, the CAT activity was 12.39% higher in the RP group, but no significant difference in CAT activity was found between the RP and CAS groups (P > 0.05). These findings, consistent with the observation of T-AOC (Fig. 1C), further indicate that RP has antioxidative effects in growing rats fed with cholesterol-free diets.
GSH content (µmol/g liver)
A
8
*
6 4 2 0 CAS
RP
GSSG content (µmol/g liver)
B
0.9
* 0.6
0.3
0 CAS
GSSG/GSH
C
RP
0.2 0.15
* 0.1 0.05 0 CAS
RP
Fig. 2. The effects of dietary proteins on hepatic reduced glutathione (A), hepatic accumulation of oxidized glutathione (B) and the ratio of GSSG/GSH (C) in 7-week-old male Wistar rats fed cholesterol-free diets for 3 weeks. The values shown represent the means ± SEM (n = 6). Bars marked with ⁎ are significantly different (P b 0.05). CAS, casein; GSH, reduced glutathione; GSSG, oxidized glutathione; RP, rice protein.
After 3 weeks of feeding, RP exhibited regulatory effects on the activities of hepatic enzymes involved in glutathione metabolism. The GST, γ-GCS and GR activities were significantly stimulated by RP feeding, by 21.57%, 65.35% and 69.28%, respectively (P b 0.05). As an important regulator in glutathione metabolism, the activity of hepatic GSHPx was 11.20% higher in rats fed RP than those fed CAS. However, a non-significant difference in GSHPx activity was observed in the CAS and RP groups under the present experimental condition (P > 0.05). Relative mRNA levels of GCLC and GCLM in livers As illustrated in Fig. 3, the mRNA levels of GCLC (Fig. 3A) and GCLM (Fig. 3B) were significantly affected by the dietary proteins. In the liver, GCLC expression was significantly higher in rats fed the RP diet than those fed the CAS diet (P b 0.05) (Fig. 3A), while RP-feeding markedly enhanced GCLM expression by 77.36% with respect to CAS (P b 0.05) (Fig. 3B). Discussion We examined the antioxidative potential of rice protein, and especially the effect of rice protein on the antioxidant defense mechanism, through the regulation of glutathione metabolism in growing rats fed cholesterol-free diets. The present study demonstrated that the lipid-lowering activity of the rice protein was closely associated with increases in the gene expression of rate-limiting enzymes in glutathione biosynthesis and the activities of hepatic antioxidative enzymes, as well as a reduction in lipid and protein oxidation, further supporting the hypothesis that hyperlipidemia is linked to accelerated oxidative stress (Leopold and Loscalzo, 2009; Roberts and Sindhu, 2009). Malondialdehyde and protein carbonyls are suggested as biomarkers of oxidative stress that may induce hypercholesterolemia (Pirinccioglu et al., 2010). The results of this study further confirmed and expended this view. After 3 weeks of feeding, lipid peroxidation and protein oxidative damage were significantly depressed in rats fed the RP diet in comparison with those fed CAS. Moreover, the findings observed in this study suggested that the decrease in hepatic MDA content by RP feeding may be a consequence of attenuated oxygen free radical-mediated damage, and the total hepatic antioxidative capacity was enhanced by RP feeding. In support of this hypothesis, there was a significant negative correlation between hepatic MDA content and hepatic T-AOC (r= −0.7094, P b 0.05). In parallel to this observation, an inhibition of hepatic PCO accumulation was also observed in growing rats fed RP. Protein carbonylation is irreversible, and PCO is a widespread indicator of severe oxidative damage and disease-derived protein dysfunction (Dalle-Donne et al., 2003). Accordingly, the accumulation of carbonylated proteins can accelerate susceptibility to reactive oxygen species (ROS)-mediated damage and induce hyperlipidemia. In support of this hypothesis, a significant positive correlation between the hepatic accumulation of PCO and plasma TC concentration (r= 0.6864, P b 0.05) was investigated in this study. Taken together, the present findings provided evidence supporting the view that increased oxidative stress products (MDA and PCO) may be the main cause of hypercholesterolemia. Additionally, to our knowledge, the present study is the first to investigate the hepatic accumulation of PCO in conjunction with cholesterol metabolism in rats fed RP. It has been demonstrated that oxidative stress results from an imbalance between oxidant and antioxidant mechanisms. Thus, the mechanisms that are most frequently suggested to be responsible for the antioxidative effects of dietary protein are the inhibition of oxidative stress and the stimulation of antioxidative defense (Fang et al., 2002; Francini et al., 2010). In this study, RP not only significantly depressed lipid and protein oxidation but also effectively enhanced the antioxidant defense mechanisms by modulating hepatic glutathione metabolism. GSH is endogenously synthesized in the liver and is
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Table 3 Effects of dietary proteins on antioxidant enzymes and glutathione metabolizing enzymes in rat liver.a
T-SOD (units/min per mg protein) CAT (units/min per mg protein) GSHPx (nmol of GSH consumed/min per mg protein) γ-GCS (μmol Pi/min per mg protein) GST (μmol of GSH consumed/min per mg protein) GR (nmol of NADPH oxidized/min per mg protein)
CAS
RP
27.27 ± 1.30 387.58 ± 18.77 306.01 ± 10.16 7.62 ± 0.44 23.69 ± 1.46 30.50 ± 1.68
43.65 ± 2.59⁎ 435.59 ± 23.52 340.27 ± 20.77 12.60 ± 0.61⁎ 28.80 ± 1.53⁎ 51.63 ± 2.17⁎
Values within the same row with ⁎ are significantly different (P b 0.05). a Values are means ± SEM for six rats per group.
the first line of defense against oxidative stress (Anderson, 1998; Lima et al., 2006; Wu et al., 2004). In general, increased levels of GSH are associated with increased tolerance to oxidative stress, leading to a decreased GSSG/GSH ratio, which is a potent indicator of tissue oxidative stress. In the light of this view, the fact that the formation and accumulation of GSSG were effectively inhibited by RP feeding under the present experimental conditions suggests that the endogenous GSH pool slowly shifted toward the oxidized state in RP group compared to the CAS group, developing an antioxidative status in the RP-fed rats. The maintenance of GSH homeostasis is regulated by several well-known enzymes. Notably, hepatic GSH content is not constant and depends on the balance between synthesis (by γ-GCS), conjugation (by GST), oxidation (by GSHPx) and the reduction of GSSG to GSH (by GR). Accordingly, the higher GSH content observed in the RP group was consistent with the following additional observations: (1) an increase in GR activity stimulated the reduction of GSSG to GSH; (2) the enzymatic activation of γ-GCS was responsible for the restoration of GSH depletion; (3) an enhancement of GST activity resulted in detoxification to decrease the oxidizing potential; and (4) increased GSHPx activity occurred due to the lack of GSH depletion. Consequently, these reactions inhibited the depletion of the
Relative GCLC mRNA level
A
1.8
*
1.5 1.2 0.9 0.6 0.3 0.0 CAS
Relative GCLM mRNA level
B
RP
2.5
*
2.0 1.5 1.0 0.5 0.0 CAS
RP
Fig. 3. The effects of dietary proteins on the hepatic expression of the glutamate cysteine ligase catalytic subunit (A) and glutamate cysteine ligase modulatory subunit genes (B) in 7-week-old male Wistar rats fed cholesterol-free diets for 3 weeks. The values shown represent the relative mRNA levels. The relative mRNA expression level in the CAS group was set to 1.00. Values are the means±SEM (n=6). Bars marked with ⁎ are significantly different (Pb 0.05). CAS, casein; GCLC, glutamate cysteine ligase catalytic subunit; GCLM, glutamate cysteine ligase modulatory subunit; RP, rice protein.
hepatic GSH pool, achieving a lower GSSG/GSH ratio in the liver of the RP group. Thus, the lipid-lowering effect was induced by the stronger antioxidative activity in the RP group. In support of this model, significant negative correlations between hepatic GSH content and plasma TC (r = − 0.9186, P b 0.05) and plasma TG (r = − 0.7053, P b 0.05) were observed in this study. On the other hand, these results also provide a mechanistic explanation for the increase in GSH level by stimulating the expression of GCLC or GCLM, which plays a major role in the rate-limiting step in the de novo GSH biosynthesis pathway (Anderson, 1998; Wu et al., 2004). Apparently, such a mechanism is required for the activation of γ-GCS. Consistent with this observation, it has been demonstrated that the overexpression of GCLC or GCLM can boost GSH biosynthesis, whereas the inhibition of GCL activity can result in a decreased GSH level and an enhanced susceptibility to oxidative stress (Forman and Dickinson, 2003). Thus, the results obtained in this study clearly suggest that rice protein may possess a vital function in improving oxidative stress and cholesterol metabolism by regulating glutathione metabolism. Similar to the antioxidant defense system in GSH, it was evident that the activities of CAT and T-SOD were also stimulated by RP in this study. More importantly, significant negative correlations between plasma cholesterol concentration and CAT activity (r= −0.7710, P b 0.05), and SOD activity (r= −0.8218, P b 0.05) were found in this study, further supporting our hypothesis that there was a link between antioxidant status and the lipid-lowering activity of RP. CAT is an important enzyme responsible for the removal of H2O2 produced under various stress conditions that protects cells from oxidative-stress-related damage, while SOD plays an important role in protecting against the toxic effects of superoxide radicals by catalyzing radical dismutation reactions. As discussed previously, GSHPx, CAT and SOD comprise the initial defense against ROS, and a decrease in their activities contributes to the oxidative insult to the tissue (Fang et al., 2002). On the other hand, CAT and GSHPx seem to have complementary catalytic activity. In view of these findings, the observed increase in GSHPx activity in the RP group, which was induced by the inhibition of GSH depletion and depressing the accumulation of H2O2, might lead to a simultaneous increase of CAT in the livers of rats fed a diet containing RP. Interestingly, however, neither GSHPx nor CAT exhibited a pronounced difference in activity between the experimental groups. At present, we cannot fully explain this phenomenon. Nevertheless, an improved understanding of the mechanism underlying the cooperation of GSHPx, CAT and SOD with respect to antioxidative status is important, suggesting that the antioxidative stress response might involve the synergistic action of various antioxidative enzymes rather than a single antioxidative reaction. Clearly, the precise mechanism remains to be clarified. Indeed, the causes of resistance to oxidative stress, which might in turn underlie the lipid-lowering effects of rice protein, appear to be multi-factorial. It has been suggested that the biological utilization of a protein is primarily dependent on its amino acid profile (Wu, 2009), providing the insight that the amino acid composition of rice protein may be a major factor in its influence on lipid metabolism, which might affect the oxidative/antioxidative status. To date, the precise mechanism by which RP improves oxidative stress has not
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been fully established, but the mechanism responsible for the effects of sulfur amino acids (SAAs) on oxidative stress should be taken into account. Recent research has demonstrated that SAAs play a significant role that is distinct from that of other amino acids by controlling oxidative stress, illustrating that SAAs are involved in the synthesis of intracellular antioxidants (e.g., GSH, taurine, etc.) and in the methionine sulfoxide reductase antioxidant system (Métayer et al., 2008; Tesseraud et al., 2009). Consistent with this hypothesis, Moundras et al. (1995) reported that methionine deficiency in rats fed soy protein could induce hypercholesterolemia and potentiates lipoprotein susceptibility to peroxidation, suggesting that methionine plays an important role in ameliorating stress conditions. Further, Yang et al. (2011b) indicated that the addition of methionine to rice protein could produce hypocholesterolemia in rats fed cholesterol-free diets. Moreover, it is confirmed that GSH synthesis is critically regulated by γ-GCS through the availability of L-cysteine (Anderson, 1998). Taken together, these findings strongly suggest that SAAs (methionine and L-cysteine) are essential compounds for the defense against oxidative stress. Thus, the higher content of SAAs (RP, 43.0 μg/mg; CAS, 32.5 μg/mg) in rice protein might represent a possible mechanism through which RP enhances oxidative stress, primarily through the modulation of GSH metabolism, further highlighting the key role of SAAs in the antioxidant status. However, additional studies are required to confirm this view. Conclusion The present study demonstrates that rice protein can improve oxidative stress, leading to an enhancement of the antioxidative defense mechanism. The lipid-lowering activity induced by rice protein is attributed in part to its stimulation of antioxidative enzyme activities and consequent reduction of the oxidative damage to lipid and protein. These results suggest that rice protein can prevent hyperlipidemia through the modification of glutathione metabolism, in which sulfur amino acids may be the main modulator responsible for antioxidative and lipid-lowering actions. The precise mechanisms involved in the antioxidative responses to rice protein await more detailed investigation. Conflict of interest statement There are no conflicts of interest to report.
Acknowledgments This work was supported in part by the National Natural Science Foundation of China (31071526), National Natural Science Foundation of Heilongjiang Province (C201027), Science Innovation Foundation of Harbin (2009RFLXN013), China Postdoctoral Science Foundation Funded Project (200902391, 20080440864) and Heilongjiang Postdoctoral Fund (LBH-Z08143). References Anderson ME. Glutathione: an overview of biosynthesis and modulation. Chem Biol Interact 1998;111–112:1-14. Avula CPR, Fernandes G. Modulation of lipid peroxidation and antioxidant enzymes in murine salivary gland by dietary fatty acid ethyl esters. Life Sci 1999;65:2373–83.
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Rice protein improves oxidative stress by regulating glutathione metabolism and attenuating oxidative damage to lipids and proteins in rats Lin Yang a,⁎, Jia-Hou Chen b, Tong Xu c, Ai-Shen Zhou b, Hong-Kun Yang b a b c
School of Food Science and Engineering, Harbin Institute of Technology, 73 Huanghe Road, Harbin 150090, China Heilongjiang Provincial Environmental Monitoring Central Station, Harbin 150056, China Heilongjiang Provincial Hospital, Harbin 150036, China
a r t i c l e
i n f o
Article history: Received 3 January 2012 Accepted 2 August 2012 Keywords: Rice protein Oxidative Antioxidative Glutathione Lipid Rats
a b s t r a c t Aims: To evaluate the effects of rice protein (RP) on glutathione metabolism and oxidative damage. Main methods: Seven-week-old male Wistar rats were fed diets containing casein and RP without cholesterol for 3 weeks. Plasma and liver lipid levels, hepatic accumulation of total glutathione (T-GSH), oxidized glutathione (GSSG), reduced glutathione (GSH), malondialdehyde (MDA) and protein carbonyl (PCO) were measured. In the liver, the total antioxidative capacity (T-AOC), mRNA levels of glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modulatory subunit (GCLM), and the activities of hepatic catalase (CAT), total superoxide dismutase (T-SOD), γ-glutamylcysteine synthetase (γ-GCS), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GSHPx) were also measured. Key findings: T-AOC, GCLC and GCLM mRNA levels, antioxidative enzyme activities (T-SOD and CAT) and glutathione metabolism related enzyme activities (γ-GCS, GST, GR and GSHPx) were effectively stimulated by RP feeding compared to casein, and RP significantly reduced the hepatic accumulation of MDA and PCO in rats. These results indicate that lipid-lowering activity was induced by RP feeding. Significance: The present study demonstrates that RP improves oxidative stress primarily through enzymatic and non-enzymatic antioxidative defense mechanisms, reflected by enhancing the antioxidative status and attenuating the oxidative damage to lipids and proteins. These results suggest that RP can prevent hyperlipidemia in part through modifying glutathione metabolism, and sulfur amino acids may be the main modulator of this antioxidative mechanism. © 2012 Elsevier Inc. All rights reserved.
Introduction Oxidative stress is one of the major risk factors for developing hyperlipidemia. Diet plays an important role in regulating oxidative stress to prevent hyperlipidemia (Avula and Fernandes, 1999; Coyle et al., 2008; Gorinstein et al., 2005; Lee, 2006). Accordingly, the suppression of oxidative stress may be a useful target for new therapies in preventing hyperlipidemia. Oxidative stress can be caused by oxidative damage to lipids and proteins. In addition to malondialdehyde (MDA), which is generally used as a marker of lipid peroxidation, the oxidation process may be accelerated by the formation and accumulation of carbonylated protein (Pirinccioglu et al., 2010). Recent studies have shown that protein carbonylation may be involved in various disease states, and protein carbonyls (PCO) may serve as biomarkers of oxidative stress (Dalle-Donne et al., 2003). Thus, lipid peroxidation and protein ⁎ Corresponding author at: Department of Food Science, School of Food Science and Engineering, Harbin Institute of Technology, 73 Huanghe Road, Nangang District, Harbin 150090, China. Tel.: +86 451 8628 2910; fax: +86 451 8628 2906. E-mail address: [email protected] (L. Yang). 0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2012.08.003
oxidative damage may indicate a higher risk for developing hyperlipidemia, suggesting that effective dietary antioxidants should be not only reactive oxygen species scavengers but also reactive carbonyl scavengers. Glutathione (GSH) is an antioxidant that is ubiquitous in mammals, and it plays important roles in several detoxification reactions and in the suppression of lipid peroxidation (Anderson, 1998). The liver represents the major site of GSH metabolism, in which GSH and its related enzymes comprise an antioxidant defense system that protects against oxidative stress. Further, as a non-essential nutrient, GSH can be synthesized in the body from several amino acids (L-cysteine, L-glutamic acid and glycine), suggesting that the amino acid profile of the diet may be a major contributor to GSH metabolism responsible for an antioxidative defense. Rice is a staple cereal that is widely consumed around the world (Kishine et al., 2008; Ohtsubo and Nakamura, 2007; Tran et al., 2005). There has been a growing emphasis on the improvement of the physiological functions of rice (Tran et al., 2004; Yang et al., 2012a), and an association between rice protein consumption and reduced plasma and liver lipid levels has been extensively demonstrated in some studies (Yang et al., 2007, 2012b; Yang and Kadowaki, 2009). However, the
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precise mechanism by which rice protein affects lipid metabolism has not been fully established. Specifically, there is not yet a comprehensive understanding of the link between the modulation of oxidative status and the consumption of rice protein. Accordingly, evidence describing how rice protein can improve oxidative stress to regulate lipid metabolism is lacking. The present study, therefore, was conducted to focus on the regulatory effects of rice protein on GSH metabolism and lipid and protein oxidative damage status. The key questions addressed are: (1) whether there is a link between lipid-lowering action and antioxidative stress and the consumption of rice protein and (2) whether rice protein can improve oxidative stress in growing rats fed cholesterol-free diets.
At the end of the feeding period, the rats were deprived of food for 18 h and then sacrificed. Blood was withdrawn from the abdominal vein into a heparinized syringe under anesthesia with sodium pentobarbital (50 mg/kg body weight), immediately cooled on ice and separated by centrifugation at 12,000 ×g for 5 min. The plasma obtained was frozen at − 20 °C until analysis. After blood collection, the liver was excised immediately, rinsed in saline and weighed after blotting on filter paper. The whole liver was cut into several portions and quickly freeze-clamped in liquid nitrogen and stored at − 80 °C until analysis.
Materials and methods
Plasma free fatty acid (FFA), triglyceride (TG), total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) concentrations were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Plasma non-high-density lipoprotein cholesterol (non-HDL-C) was calculated as non-HDL-C = TC − HDL-C. The lipids in the liver were extracted and purified as described by Folch et al. (1957) and analyzed as described by Yang et al. (2011a). Samples of liver were extracted with chloroform/methanol (2:1, v/v). Hepatic total cholesterol, triglyceride and free fatty acid levels were measured with a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Protein sources Rice protein (RP) from Oryza sativa L. cv. Longjing 26 (Rice Research Institute of Heilongjiang Academy of Agricultural Sciences, Jiamusi, China) and casein (CAS) (Gansu Hualing Industrial Group, Gansu, China) were used as the dietary protein sources in the present study. RP was prepared by the alkaline extraction method (Yang et al., 2011a, 2012b). Animals and diets The present experiment was performed in compliance with the Guidelines of the Committee for Animal Experimentation of Harbin Medical University and followed the same protocol used in previous studies (Yang et al., 2011a, 2012b). Seven-week-old male Wistar rats were purchased from the Animal Center of Harbin Medical University (Harbin, China) and individually housed in metabolic cages in a room maintained at 22 ± 2 °C under a 12 h light–dark cycle (07:00–19:00 light). Rats were allowed free access to commercial pellets (Animal Center of Harbin Medical University, Harbin, China) for 3 days. After acclimatization, the rats were randomly divided into two groups of similar body weight. Each group consisted of six rats. All animals were fed ad libitum with experimental diets according to the formula recommended by the American Institute of Nutrition (Reeves et al., 1993). For 3 weeks, growing rats were fed cholesterolfree diets with a dietary protein level of 20% (as crude protein, CP) of CAS and RP, respectively. Diets were completed to 100% with starch. The compositions of the experimental diets are shown in Table 1.
Analyses of plasma and liver lipid levels
Determination of malondialdehyde and protein carbonyl The hepatic malondialdehyde (MDA) and protein carbonyl (PCO) contents were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Estimation of hepatic total antioxidative capacity The hepatic total antioxidative capacity (T-AOC) was measured with a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Measurements of reduced glutathione and oxidized glutathione Total glutathione (T-GSH) and oxidized glutathione (GSSG) in the liver were assayed with commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Reduced glutathione (GSH) was calculated as GSH = T-GSH − 2 × GSSG.
Sample collection Analyses of hepatic enzyme activities During the feeding period, food consumption and body weight were recorded daily in the morning before replenishing the diets.
Table 1 Composition of the experimental diets (g/kg diet).
CAS RP Sucrose Cellulose Soybean oil β-Cornstarch Mineral mixc Vitamin mixd Choline bitartrate Tert-butylhydroquinone L-Cysteine a b c d
CASa
RPb
228.9 – 100.0 50.0 70.0 500.6 35.0 10.0 2.5 0.014 3.0
– 221.8 100.0 50.0 70.0 510.7 35.0 10.0 2.5 0.014 –
CAS, casein, crude protein 873.7 g/kg. RP, rice protein, crude protein 901.7 g/kg. Mineral mixture, AIN-93G-MX (Nosan Corp., Japan). Vitamin mixture, AIN-93-VX (Nosan Corp., Japan).
The activities of hepatic total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSHPx), glutathione reductase (GR), glutathione-S-transferase (GST) and γ-glutamyl cysteine synthetase (γ-GCS) were determined using kits from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Quantitative real-time PCR Total RNA was extracted from rat livers using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. cDNA was reverse transcribed from 1 μg of total RNA using a PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Bio. Inc., Otsu, Shiga, Japan). For quantitative real time PCR, cDNA was analyzed with the ABI 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) using SYBR Green (Takara Bio. Inc., Otsu, Shiga, Japan). The primer sequences used were: 5′-ACAGCAACAGGGTGGT GG-AC-3′ (forward) and 5′-TTTGAGGGTGCAGCGAACTT-3′ (reverse) for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH); 5′-CC TCCTCCTCCAAACTCAGAT-A-3′ (forward) and 5′-CCACAAATACCACAT
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AGGCAGA-3′ (reverse) for rat glutamate cysteine ligase catalytic subunit (GCLC); 5′-GGGCACAGGTAAAACCCAATA-3′ (forward) and 5′-TTCAATGTCAGGGATGCTTTC-3′ (reverse) for rat glutamate cysteine ligase modulatory subunit (GCLM). The results were normalized to the level of GAPDH mRNA. Statistical analysis The data are presented as the means ± SEM. Differences between groups were examined for statistical significance using one-way analysis of variance (ANOVA) and then determined with the least significant difference test. The criterion for significance was P b 0.05.
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different from that in the CAS group (P > 0.05). Like the TG concentration, the plasma FFA level was also 14.47% lower in the RP group than in the CAS group (P > 0.05). The hepatic total cholesterol concentrations were significantly lower in rats fed RP than those fed CAS (P b 0.05) (Table 2), consistent with previous studies (Yang et al., 2007, 2011a). In parallel, hepatic triglyceride levels decreased by 35.22% in RP compared to CAS (P b 0.05). As shown in Table 2, the significant reduction in hepatic triglyceride deposits was mainly reflected by the diminished concentrations of free fatty acids in the RP group. However, no significant difference in free fatty acid concentration was found between the CAS and RP groups (P > 0.05), although RP feeding reduced hepatic free fatty acid levels by 13.02%.
Results Malondialdehyde and protein carbonyls Body weight, food intake and liver weight Body weight gain was significantly reduced in rats fed RP compared with CAS (P b 0.05) (Table 2). No significant change in food intake was found between RP and CAS, suggesting that the dietary protein did not affect food intake during the growing period. As shown in Table 2, less liver swelling was observed in rats fed RP (P b 0.05) than in those fed CAS, as suggested by previous studies (Yang et al., 2007, 2011a).
Total antioxidative capacity
CAS
RP
21.33 ± 0.63 5.28 ± 0.24 8.85 ± 0.33
19.98 ± 0.35 4.27 ± 0.26⁎ 7.83 ± 0.26⁎
1.51 ± 0.03 0.93 ± 0.06 0.58 ± 0.05 0.62 ± 0.09 0.41 ± 0.03 0.44 ± 0.03 0.76 ± 0.03
1.34 ± 0.02⁎ 0.95 ± 0.05 0.39 ± 0.04⁎ 0.41 ± 0.06⁎
15.07 ± 0.50 58.49 ± 2.21 29.73 ± 1.36
11.00 ± 0.44⁎ 37.89 ± 1.96⁎ 25.86 ± 1.14
0.35 ± 0.02 0.37 ± 0.03 0.65 ± 0.03
Values within the same row with ⁎ are significantly different (P b 0.05). a Values are means ± SEM for six rats per group.
5 4
*
3 2 1 0 CAS
B Protein carbonyls contents (nmol/mg protein)
Table 2 Food intake, body weight gain, liver weight, plasma and liver lipids of rats fed experimental diets.a
A
C
RP
4
*
3 2 1 0 CAS
T-AOC (units/min per mg protein)
As shown in Table 2, RP significantly reduced the plasma TC concentration, by 11.26%, compared with CAS (P b 0.05), showing that RP exhibited an effective hypocholesterolemic activity in growing rats. Concomitant with the decrease in plasma TC, the plasma non-HDL-C concentration was also distinctly lower in rats fed RP than in those fed CAS (P b 0.05), whereas the HDL-C level did not differ significantly between experimental groups (P > 0.05). As a result, the ratio of non-HDL-C/HDL-C was significantly decreased, by 33.87%, in the RP group compared with the CAS group (P b 0.05). The results indicated that RP-feeding effectively altered the cholesterol distribution, reflected mainly by the lower concentration of non-HDL-C in the RP group. Accompanying the decreased level of TC, the plasma TG concentration was also reduced by RP feeding in this study, compared with CAS (Table 2). However, no significant difference in TG level was observed between the CAS and RP groups, despite the fact that RP feeding lowered the TG by 14.63% (P > 0.05). The TG to HDL ratio also decreased in the RP group (− 15.91%) but was not significantly
MDA contents (nmol/mg protein)
As shown in Fig. 1C, hepatic total antioxidative capacity (T-AOC) was significantly stimulated by RP in growing rats fed cholesterol-free diets
Plasma lipids, lipoprotein profiles and hepatic lipids
Food intake (g/day) Body weight gain (g/day) Liver weight (g) Plasma lipids (mmol/L) Cholesterol TC HDL-C Non-HDL-C Non-HDL-C/HDL-C (mol/mol) Triglyceride TG/HDL-C (mol/mol) Free fatty acids Hepatic lipids (μmol/g liver) Cholesterol Triglyceride Free fatty acids
After 3 weeks of feeding, the hepatic accumulations of MDA (Fig. 1A) and PCO (Fig. 1B) were significantly altered by the dietary protein treatments, showing a marked difference in the oxidative damage to lipid and protein between CAS and RP. The MDA and PCO contents were significantly decreased when rats were fed an RP diet (MDA: − 23.02%; PCO: − 21.34%) (P b 0.05).
2
RP
*
1.6 1.2 0.8 0.4 0 CAS
RP
Fig. 1. The effects of dietary proteins on the hepatic contents of lipid peroxidation products (A), protein carbonyls (B) and hepatic total antioxidative capacity (C) in 7-week-old male Wistar rats fed cholesterol-free diets for 3 weeks. The values shown represent the means ± SEM (n = 6). Bars marked with ⁎ are significantly different (P b 0.05). CAS, casein; MDA, malondialdehyde; RP, rice protein; T-AOC, total antioxidative capacity.
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(P b 0.05). Compared with CAS, RP feeding produced a marked increase in T-AOC (27.54%), suggesting that RP feeding could stimulate the antioxidative status under the present experimental conditions.
Glutathione contents As illustrated in Fig. 2, hepatic glutathione contents were effectively regulated by dietary proteins. Compared with CAS, the hepatic concentration of GSH (Fig. 2A) was increased 40.91% by RP feeding (P b 0.05), whereas the accumulation of GSSG in the liver was depressed by 18.06% in the RP group (Fig. 2B) (P b 0.05). As a result, a pronounced reduction in the ratio of GSSG to GSH was produced by RP compared to CAS (Fig. 2C) (P b 0.05).
Hepatic enzyme activities As summarized in Table 3, the activities of various hepatic enzymes involved in antioxidative activities and glutathione metabolism were affected by the dietary protein. Compared with CAS, T-SOD was enhanced 60.07% by RP feeding (P b 0.05). Similarly, the CAT activity was 12.39% higher in the RP group, but no significant difference in CAT activity was found between the RP and CAS groups (P > 0.05). These findings, consistent with the observation of T-AOC (Fig. 1C), further indicate that RP has antioxidative effects in growing rats fed with cholesterol-free diets.
GSH content (µmol/g liver)
A
8
*
6 4 2 0 CAS
RP
GSSG content (µmol/g liver)
B
0.9
* 0.6
0.3
0 CAS
GSSG/GSH
C
RP
0.2 0.15
* 0.1 0.05 0 CAS
RP
Fig. 2. The effects of dietary proteins on hepatic reduced glutathione (A), hepatic accumulation of oxidized glutathione (B) and the ratio of GSSG/GSH (C) in 7-week-old male Wistar rats fed cholesterol-free diets for 3 weeks. The values shown represent the means ± SEM (n = 6). Bars marked with ⁎ are significantly different (P b 0.05). CAS, casein; GSH, reduced glutathione; GSSG, oxidized glutathione; RP, rice protein.
After 3 weeks of feeding, RP exhibited regulatory effects on the activities of hepatic enzymes involved in glutathione metabolism. The GST, γ-GCS and GR activities were significantly stimulated by RP feeding, by 21.57%, 65.35% and 69.28%, respectively (P b 0.05). As an important regulator in glutathione metabolism, the activity of hepatic GSHPx was 11.20% higher in rats fed RP than those fed CAS. However, a non-significant difference in GSHPx activity was observed in the CAS and RP groups under the present experimental condition (P > 0.05). Relative mRNA levels of GCLC and GCLM in livers As illustrated in Fig. 3, the mRNA levels of GCLC (Fig. 3A) and GCLM (Fig. 3B) were significantly affected by the dietary proteins. In the liver, GCLC expression was significantly higher in rats fed the RP diet than those fed the CAS diet (P b 0.05) (Fig. 3A), while RP-feeding markedly enhanced GCLM expression by 77.36% with respect to CAS (P b 0.05) (Fig. 3B). Discussion We examined the antioxidative potential of rice protein, and especially the effect of rice protein on the antioxidant defense mechanism, through the regulation of glutathione metabolism in growing rats fed cholesterol-free diets. The present study demonstrated that the lipid-lowering activity of the rice protein was closely associated with increases in the gene expression of rate-limiting enzymes in glutathione biosynthesis and the activities of hepatic antioxidative enzymes, as well as a reduction in lipid and protein oxidation, further supporting the hypothesis that hyperlipidemia is linked to accelerated oxidative stress (Leopold and Loscalzo, 2009; Roberts and Sindhu, 2009). Malondialdehyde and protein carbonyls are suggested as biomarkers of oxidative stress that may induce hypercholesterolemia (Pirinccioglu et al., 2010). The results of this study further confirmed and expended this view. After 3 weeks of feeding, lipid peroxidation and protein oxidative damage were significantly depressed in rats fed the RP diet in comparison with those fed CAS. Moreover, the findings observed in this study suggested that the decrease in hepatic MDA content by RP feeding may be a consequence of attenuated oxygen free radical-mediated damage, and the total hepatic antioxidative capacity was enhanced by RP feeding. In support of this hypothesis, there was a significant negative correlation between hepatic MDA content and hepatic T-AOC (r= −0.7094, P b 0.05). In parallel to this observation, an inhibition of hepatic PCO accumulation was also observed in growing rats fed RP. Protein carbonylation is irreversible, and PCO is a widespread indicator of severe oxidative damage and disease-derived protein dysfunction (Dalle-Donne et al., 2003). Accordingly, the accumulation of carbonylated proteins can accelerate susceptibility to reactive oxygen species (ROS)-mediated damage and induce hyperlipidemia. In support of this hypothesis, a significant positive correlation between the hepatic accumulation of PCO and plasma TC concentration (r= 0.6864, P b 0.05) was investigated in this study. Taken together, the present findings provided evidence supporting the view that increased oxidative stress products (MDA and PCO) may be the main cause of hypercholesterolemia. Additionally, to our knowledge, the present study is the first to investigate the hepatic accumulation of PCO in conjunction with cholesterol metabolism in rats fed RP. It has been demonstrated that oxidative stress results from an imbalance between oxidant and antioxidant mechanisms. Thus, the mechanisms that are most frequently suggested to be responsible for the antioxidative effects of dietary protein are the inhibition of oxidative stress and the stimulation of antioxidative defense (Fang et al., 2002; Francini et al., 2010). In this study, RP not only significantly depressed lipid and protein oxidation but also effectively enhanced the antioxidant defense mechanisms by modulating hepatic glutathione metabolism. GSH is endogenously synthesized in the liver and is
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Table 3 Effects of dietary proteins on antioxidant enzymes and glutathione metabolizing enzymes in rat liver.a
T-SOD (units/min per mg protein) CAT (units/min per mg protein) GSHPx (nmol of GSH consumed/min per mg protein) γ-GCS (μmol Pi/min per mg protein) GST (μmol of GSH consumed/min per mg protein) GR (nmol of NADPH oxidized/min per mg protein)
CAS
RP
27.27 ± 1.30 387.58 ± 18.77 306.01 ± 10.16 7.62 ± 0.44 23.69 ± 1.46 30.50 ± 1.68
43.65 ± 2.59⁎ 435.59 ± 23.52 340.27 ± 20.77 12.60 ± 0.61⁎ 28.80 ± 1.53⁎ 51.63 ± 2.17⁎
Values within the same row with ⁎ are significantly different (P b 0.05). a Values are means ± SEM for six rats per group.
the first line of defense against oxidative stress (Anderson, 1998; Lima et al., 2006; Wu et al., 2004). In general, increased levels of GSH are associated with increased tolerance to oxidative stress, leading to a decreased GSSG/GSH ratio, which is a potent indicator of tissue oxidative stress. In the light of this view, the fact that the formation and accumulation of GSSG were effectively inhibited by RP feeding under the present experimental conditions suggests that the endogenous GSH pool slowly shifted toward the oxidized state in RP group compared to the CAS group, developing an antioxidative status in the RP-fed rats. The maintenance of GSH homeostasis is regulated by several well-known enzymes. Notably, hepatic GSH content is not constant and depends on the balance between synthesis (by γ-GCS), conjugation (by GST), oxidation (by GSHPx) and the reduction of GSSG to GSH (by GR). Accordingly, the higher GSH content observed in the RP group was consistent with the following additional observations: (1) an increase in GR activity stimulated the reduction of GSSG to GSH; (2) the enzymatic activation of γ-GCS was responsible for the restoration of GSH depletion; (3) an enhancement of GST activity resulted in detoxification to decrease the oxidizing potential; and (4) increased GSHPx activity occurred due to the lack of GSH depletion. Consequently, these reactions inhibited the depletion of the
Relative GCLC mRNA level
A
1.8
*
1.5 1.2 0.9 0.6 0.3 0.0 CAS
Relative GCLM mRNA level
B
RP
2.5
*
2.0 1.5 1.0 0.5 0.0 CAS
RP
Fig. 3. The effects of dietary proteins on the hepatic expression of the glutamate cysteine ligase catalytic subunit (A) and glutamate cysteine ligase modulatory subunit genes (B) in 7-week-old male Wistar rats fed cholesterol-free diets for 3 weeks. The values shown represent the relative mRNA levels. The relative mRNA expression level in the CAS group was set to 1.00. Values are the means±SEM (n=6). Bars marked with ⁎ are significantly different (Pb 0.05). CAS, casein; GCLC, glutamate cysteine ligase catalytic subunit; GCLM, glutamate cysteine ligase modulatory subunit; RP, rice protein.
hepatic GSH pool, achieving a lower GSSG/GSH ratio in the liver of the RP group. Thus, the lipid-lowering effect was induced by the stronger antioxidative activity in the RP group. In support of this model, significant negative correlations between hepatic GSH content and plasma TC (r = − 0.9186, P b 0.05) and plasma TG (r = − 0.7053, P b 0.05) were observed in this study. On the other hand, these results also provide a mechanistic explanation for the increase in GSH level by stimulating the expression of GCLC or GCLM, which plays a major role in the rate-limiting step in the de novo GSH biosynthesis pathway (Anderson, 1998; Wu et al., 2004). Apparently, such a mechanism is required for the activation of γ-GCS. Consistent with this observation, it has been demonstrated that the overexpression of GCLC or GCLM can boost GSH biosynthesis, whereas the inhibition of GCL activity can result in a decreased GSH level and an enhanced susceptibility to oxidative stress (Forman and Dickinson, 2003). Thus, the results obtained in this study clearly suggest that rice protein may possess a vital function in improving oxidative stress and cholesterol metabolism by regulating glutathione metabolism. Similar to the antioxidant defense system in GSH, it was evident that the activities of CAT and T-SOD were also stimulated by RP in this study. More importantly, significant negative correlations between plasma cholesterol concentration and CAT activity (r= −0.7710, P b 0.05), and SOD activity (r= −0.8218, P b 0.05) were found in this study, further supporting our hypothesis that there was a link between antioxidant status and the lipid-lowering activity of RP. CAT is an important enzyme responsible for the removal of H2O2 produced under various stress conditions that protects cells from oxidative-stress-related damage, while SOD plays an important role in protecting against the toxic effects of superoxide radicals by catalyzing radical dismutation reactions. As discussed previously, GSHPx, CAT and SOD comprise the initial defense against ROS, and a decrease in their activities contributes to the oxidative insult to the tissue (Fang et al., 2002). On the other hand, CAT and GSHPx seem to have complementary catalytic activity. In view of these findings, the observed increase in GSHPx activity in the RP group, which was induced by the inhibition of GSH depletion and depressing the accumulation of H2O2, might lead to a simultaneous increase of CAT in the livers of rats fed a diet containing RP. Interestingly, however, neither GSHPx nor CAT exhibited a pronounced difference in activity between the experimental groups. At present, we cannot fully explain this phenomenon. Nevertheless, an improved understanding of the mechanism underlying the cooperation of GSHPx, CAT and SOD with respect to antioxidative status is important, suggesting that the antioxidative stress response might involve the synergistic action of various antioxidative enzymes rather than a single antioxidative reaction. Clearly, the precise mechanism remains to be clarified. Indeed, the causes of resistance to oxidative stress, which might in turn underlie the lipid-lowering effects of rice protein, appear to be multi-factorial. It has been suggested that the biological utilization of a protein is primarily dependent on its amino acid profile (Wu, 2009), providing the insight that the amino acid composition of rice protein may be a major factor in its influence on lipid metabolism, which might affect the oxidative/antioxidative status. To date, the precise mechanism by which RP improves oxidative stress has not
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been fully established, but the mechanism responsible for the effects of sulfur amino acids (SAAs) on oxidative stress should be taken into account. Recent research has demonstrated that SAAs play a significant role that is distinct from that of other amino acids by controlling oxidative stress, illustrating that SAAs are involved in the synthesis of intracellular antioxidants (e.g., GSH, taurine, etc.) and in the methionine sulfoxide reductase antioxidant system (Métayer et al., 2008; Tesseraud et al., 2009). Consistent with this hypothesis, Moundras et al. (1995) reported that methionine deficiency in rats fed soy protein could induce hypercholesterolemia and potentiates lipoprotein susceptibility to peroxidation, suggesting that methionine plays an important role in ameliorating stress conditions. Further, Yang et al. (2011b) indicated that the addition of methionine to rice protein could produce hypocholesterolemia in rats fed cholesterol-free diets. Moreover, it is confirmed that GSH synthesis is critically regulated by γ-GCS through the availability of L-cysteine (Anderson, 1998). Taken together, these findings strongly suggest that SAAs (methionine and L-cysteine) are essential compounds for the defense against oxidative stress. Thus, the higher content of SAAs (RP, 43.0 μg/mg; CAS, 32.5 μg/mg) in rice protein might represent a possible mechanism through which RP enhances oxidative stress, primarily through the modulation of GSH metabolism, further highlighting the key role of SAAs in the antioxidant status. However, additional studies are required to confirm this view. Conclusion The present study demonstrates that rice protein can improve oxidative stress, leading to an enhancement of the antioxidative defense mechanism. The lipid-lowering activity induced by rice protein is attributed in part to its stimulation of antioxidative enzyme activities and consequent reduction of the oxidative damage to lipid and protein. These results suggest that rice protein can prevent hyperlipidemia through the modification of glutathione metabolism, in which sulfur amino acids may be the main modulator responsible for antioxidative and lipid-lowering actions. The precise mechanisms involved in the antioxidative responses to rice protein await more detailed investigation. Conflict of interest statement There are no conflicts of interest to report.
Acknowledgments This work was supported in part by the National Natural Science Foundation of China (31071526), National Natural Science Foundation of Heilongjiang Province (C201027), Science Innovation Foundation of Harbin (2009RFLXN013), China Postdoctoral Science Foundation Funded Project (200902391, 20080440864) and Heilongjiang Postdoctoral Fund (LBH-Z08143). References Anderson ME. Glutathione: an overview of biosynthesis and modulation. Chem Biol Interact 1998;111–112:1-14. Avula CPR, Fernandes G. Modulation of lipid peroxidation and antioxidant enzymes in murine salivary gland by dietary fatty acid ethyl esters. Life Sci 1999;65:2373–83.
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