antioxidant enzyme assays

6) 1578–1584www.elsevier.com/locate/lifescie

Life Sciences 79 (200

Effects of soy protein and genistein on blood glucose, antioxidant enzymeactivities, and lipid profile in streptozotocin-induced diabetic rats

Jeong-Sook Lee ⁎

Department of Food and Nutrition, Kosin University, Busan, 606-701, South Korea

Received 18 May 2006; accepted 19 June 2006

Abstract

In the current study, the effect of soy protein and genistein, one of the main isoflavones in soybeans, on blood glucose, lipid profile, andantioxidant enzyme activities in streptozotocin (STZ)-induced diabetic rats was investigated. Male Sprague–Dawley rats were divided intonondiabetic control, STZ, STZ-genistein supplemented group (STZ-G; 600 mg/kg diet), and STZ-isolated soy protein supplemented group (STZ-ISP; 200 g/kg diet). Diabetes was induced by a single injection of STZ (50 mg/kg BW) freshly dissolved in 0.1 mol/L citrate buffer (pH 4.5) intothe intraperitonium. Diabetes was confirmed by measuring the fasting blood glucose concentration 48-h post-injection. The rats with bloodglucose level above 350 mg/dL were considered to be diabetic. Genistein and ISP were supplemented in the diet for 3 weeks.

The supplementation of genistein and ISP increased the plasma insulin level but decreased the HbAIC level of the STZ-induced diabetic rats.The supplementation of genistein and ISP increased the glucokinase level of the STZ-induced diabetic rats. A significant reduction in glucose-6-phosphatase was observed in the groups treated with genistein and ISP in comparison with the diabetic control group. Hepatic superoxidedismutase, catalase, and glutathione peroxidase activities of the STZ-induced diabetic rats were significantly decreased in comparison with thecontrol rats. Administering genistein and ISP to the STZ-induced diabetic rats significantly increased those enzyme activities. The concentration ofthiobarbituric acid reactive substances in the STZ-induced diabetic rats was significantly elevated, while the genistein and ISP supplementdecreased it to the control concentration. Genistein and ISP supplements seem to be beneficial for correcting the hyperglycemia and preventingdiabetic complications.© 2006 Elsevier Inc. All rights reserved.

Keywords: Soy protein; Genistein; Streptozotocin-induced diabetic rats; Antioxidant enzymes

Introduction

Diabetes mellitus is a major endocrine disorder and growinghealth problem in most countries (Gavard et al., 1993; Andersonet al., 2001). Recently, it was suggested that formation of freeradicals is involved in the pathogenesis of diabetes and thedevelopment of diabetic complications because prolongedexposure to hyperglycemia increases the generation of freeradicals and reduces capacities of the antioxidant defense system(Sanders et al., 2001). In spite of the presentation of manyhypoglycemic agents, diabetes and its related complications arestill a major medical problem.

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0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.lfs.2006.06.030

The interest in the potential health effects of soy and soyisoflavones is growing as epidemiological studies have associatedwith a diet rich in isoflavones with a lower risk of certain diseases(Anderson et al., 1995; Potter, 1998; Hermansen et al., 2001). Soyintake has been linked to the improved blood lipid levels inhumans and animals and decreased arterial fatty streaks in ani-mals, therefore reducing the risk of developing atherosclerosis(Adams et al., 2002; Lichtenstein, 2001; Iqbal et al., 2002).Recently, isoflavones as an important bio-active component ofsoy also have been investigated (Adams et al., 2002; Clarkson,2002; Nestel, 2002).

Considerable research efforts have focused on isoflavones asthe main hypolipidemic agent in soy because of their anti-oxidative and mild estrogenic activity (Anthony, 2000; Polk-owski and Mazurek, 2000; Wilson et al., 2002). Some studieshave shown that removal of the isoflavone-containing fraction of

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1579J.-S. Lee / Life Sciences 79 (2006) 1578–1584

soy protein results in the loss of soy's beneficial effect on bloodlipids (Anthony et al., 1998; Crouse et al., 1999). It remains apossibility that soy has a positive and direct effect on the man-agement of diabetes by some yet-unrecognized mechanism.However, the interaction between soy protein and isoflavone anddiabetic complications is little known. In this study, the possibleanti-diabetic effects of soy protein and genistein, one of the mainisoflavones in soybeans, in streptozotocin-induced diabetic ratshave been evaluated.

Material and methods

Animals and diets

At the beginning of the experiment, Male Sprague–Dawleyrats weighing between 80 and 90 g were purchased fromDaehanLaboratory Animal Research Center (Daegu, Korea). The ani-mals were all individually housed in stainless steel cages in anair-conditioned room with controlled temperature (20–22 °C)and automatic lighting (alternation 12-h periods of light anddark) and fed an AIN-93 (Reeves et al., 1993) standardlaboratory diet for 21 days after arrival. The animals were di-vided into two groups: a nondiabetic control and a diabeticgroup. Diabetes was induced by a single injection of STZ(50 mg/kg BW; Sigma, USA) freshly dissolved in a 0.1 mol/Lcitrate buffer (pH 4.5) into the intraperitonium. The control ratswere only injected with the citrate buffer. Diabetes was con-firmed in the STZ-treated rats by measuring the fasting bloodglucose concentration 48-h post-injection. The rats with bloodglucose level above 350 mg/dL were considered to be diabeticand were used in the experiment. The diabetic rats wererandomly divided into three sub-groups, diabetic controls (STZ),diabetic rats given genistein (STZ-G; 600 mg/kg diet), anddiabetic rats given isolated soy protein (STZ-ISP; 200 g/kg diet).In this study, 32 rats were used (eight control and 24 diabetic).The composition of the experimental diet, as shown in Table 1,was based on the AIN-93 standard laboratory diet. The rats weregiven free access to food and distilled water, and all animals wereobserved daily for any clinical signs of disease.

Table 1Composition of control and experimental diet (g/100 g diet)

Component Control STZ-G STZ-ISP

Caseina 20.0 20.0 –Isolated soy proteinb – – 20.0Cornstarch 55.0 54.94 55.0Sucrose 10.0 10.0 10.0Corn oil 5.0 5.0 5.0Cellulose 5.0 5.0 5.0Mineral mixturec 3.5 3.5 3.5Vitamin mixtured 1.0 1.0 1.0Choline bitartrate 0.2 0.2 0.2DL-methione 0.3 0.3 0.3Genistein – 0.06 –a Casein (ICN Biomedicals, Costa Mesa, USA).b Soy protein isolates (Protein Technologies International, St. Louis, USA).c AIN-93 mineral mixture.d AIN-93 vitamin mixture.

The food consumption and weight gain were measureddaily and weekly, respectively. At the end of the experimentalperiod (3 weeks), the rats were anesthetized with ether fol-lowing a 16-h fast. Blood samples were taken from the ab-dominal aorta using heparin-coated syringes for plasma andregular syringes for serum. Plasma and serum were obtainedby centrifuging the blood at 3000 rpm for 15 min at 4 °C. Thelivers were removed and rinsed with physiological saline. Allsamples were stored at −70 °C until analyzed.

Glucose tolerance test

After 18 days of treatment, a fasting blood sample was takenfrom all the groups of rats. Four more blood samples werecollected at 30-, 60-, 90-, and 120-min intervals after adminis-tration of glucose at a concentration of 2 g/kg of body weight(Joy and Kuttan, 1999). All the blood samples were collectedwith potassium oxalate and sodium fluoride solution for theestimation of glucose.

Tissue preparations

The livers were homogenized in 20 parts (w/v) of a 0.25mol/Lsucrose solution using a tissue homogenizer with a Teflon pestleat 4 °C. The homogenate was centrifuged at 600 ×g for 10 min todiscard any cell debris, then the supernatant was further centri-fuged at 10,000 ×g for 20 min to remove the mitochondria pellet.Finally, the supernatant was further ultracentrifuged at 105,000 ×gfor 60 min to obtain the cytosol supernatant. The amounts ofprotein in the mitochondrial and cytosolic fractions weremeasured using the method of Lowry et al. (1951) with bovineserum albumin as the standard.

Plasma insulin and glycosylated hemoglobin levels

Plasma insulin was determined by using a rat insulinradioimmunoassay kit (Linco Research Inc., St. Charles, USA)in a gamma counter (Peckard, USA) based on the method ofAndersen et al. (1993). The glycosylated hemoglobin (HbAIC)was determined using a commercial kit (Roche Co., Basel,Switzland) based on the method of Goldstein et al. (1986).

Glucokinase and glucose-6-phosphatase activities

The glucokinase activity was determined using the method ofDavidson and Arion (1987). The activity of glucose-6-phospha-tase was measured using the method of Alegre et al. (1988).

Serum and hepatic lipids

The serum total cholesterol level was determined using acommercial kit (Sigma, USA) based on a modification of thecholesterol oxidase method of Allain et al. (1974). The HDL-fractions were separated using a Sigma kit based on the heparin-manganese precipitation procedure (Warnick and Albers, 1978),and the HDL-cholesterol concentration was determined using thesame enzymatic method. The serum triglyceride concentration

Fig. 2. Plasma insulin level in experimental groups (n=8). The insulin values(mean±SD) are expressed as ng/mL. The means sharing a common letter are notsignificantly different ( p<0.05).

1580 J.-S. Lee / Life Sciences 79 (2006) 1578–1584

was measured enzymatically using a kit from Sigma ChemicalCo., a modification of the lipase-glycerol phosphate oxidasemethod (McGrowan et al., 1983). The hepatic lipids wereextracted using the procedure of Folch et al. (1957). The driedlipid residues were dissolved in 1 mL ethanol for cholesterol andtriglycerides assays. Triton X-100 and sodium cholate solutions(in distilled H2O) were added to 200 μL of the dissolved lipidsolution to produce final concentrations of 5 g/L and 3 mmol/L,respectively. The hepatic cholesterol and the triglyceride wereanalyzed with the same enzymatic kit used in the plasma analysis.

Antioxidant enzyme activities and TBARS concentration

The hepatic superoxide dismutase (SOD) activity was de-termined using Marklund and Marklund's method (Marklundand Marklund, 1974). The hepatic catalase (CAT) activity wasmeasured using Abei's method (Abei, 1984). The activity ofhepatic glutathione peroxidase (GSH-Px) was measured usingPaglia and Valentine's method (Paglia and Valentine, 1967). Thehepatic thiobarbituric acid reactive substances (TBARS) weremonitored according to the method of Tarladgis et al. (1964).

Serum aminotransferase activity and plasma urea level

The serum aspartate aminotransferase (AST) and alanine ami-notransferase (ALT) activities were determined using a commer-cial kit (EikenCo., Tokyo, Japan) based on themethod ofReitmanand Frankel (1957). Plasma concentration of urea was determinedby the methods of Patton and Crouch (1977).

Statistical analyses

All data are presented as the mean±SE. The data were eval-uated by a one-way ANOVA using the SPSS program, and thedifferences between the means assessed using Duncan's

Fig. 1. Oral glucose tolerance test in experimental groups (n=8). The bloodglucose levels (mean±SD) are expressed as mg/dL. The means sharing acommon letter are not significantly different ( p<0.05).

multiple range test. Statistical significance was considered atp<0.05.

Results

Blood glucose, plasma insulin and glycosylated hemoglobinlevels

Fig. 1 shows the blood glucose levels of control andexperimental groups of rats after oral administration of glucose.The blood glucose level in the control rats rose to a peak value30 min after glucose load and decreased to near normal levels at120 min. In diabetic control rats, the peak increase in bloodglucose concentration was observed after 30 min and remainedhigh over the 90 min. Genistein and ISP treated diabetic ratsshowed significant decreases in blood glucose level at 30 and120 min compared with diabetic rats.

Fig. 3. Glacosylated hemoglobin level in experimental groups (n=8). The gly-cosylated hemoglobin values (mean±SD) are expressed as mg/g of hemoglobin.The means sharing a common letter are not significantly different ( p<0.05).

Table 4Effect of genistein and isolated soy protein on hepatic SOD, CAT, GSH-Pxactivities and TBARS concentration in diabetic rats

Control STZ STZ-G STZ-ISP

SOD1 12.79±0.75a 6.50±0.54c 10.52±1.06b 11.00±1.12b

CAT2 18.60±1.13a 11.06±0.91d 12.41±1.13c 14.30±1.19b

GSH-Px3 3.01±0.46a 1.23±0.46c 2.16±0.46b 2.27±0.77b

TBARS4 15.12±0.14b 29.53±2.12a 19.24±1.06b 17.91±1.23b

Values are mean±SD of 8 rats from each group.a,b,c Means in the same row not sharing a common superscript are significantlydifferent ( p<0.05) between groups.1 Superoxide dismutase: units/mg protein.2 Catalase: deceased H2O2 nmol/min/mg protein.3 Glutathione peroxidase: oxidized NADPH nmol/min/mg protein.4 Thiobarbituric acid reactive substances: nmol/g.

Table 2Effect of genistein and isolated soy protein on hepatic glucokinase and glucose-6-phosphatase activities in diabetic rats


Glucokinase1 0.261±0.023a 0.079±0.009d 0.103±0.012c 0.133±0.012b

Glucose-6-phosphatase2

0.165±0.011d 0.457±0.014a 0.396±0.022b 0.302±0.013c

Values are mean±SD of 8 rats from each group.a,b,c Means in the same row not sharing a common superscript are significantlydifferent ( p<0.05) between groups.1 Glucokinase: units/h/mg of protein.2 Glucose-6-phosphatase: units/min/mg of protein.


Plasma insulin levels were lower in the STZ-induced diabeticrats compared with the control rats (Fig. 2). The supplementationof genistein and ISP increased the plasma insulin level of the STZ-induced diabetic rats. The effect was more pronounced in the ISPsupplemented rats than in the genistein-supplemented rats.

HbAIC levels were higher in the STZ-induced diabetic ratscompared to the control rats (Fig. 3). The supplementation ofgenistein and ISP decreased the HbAIC level of the STZ-induced diabetic rats. The effects were more potent in the STZ-ISP group than in the STZ-G group.

Glucokinase and glucose-6-phosphatase activities

Table 2 shows the effect of genistein and ISP on glucokinaseand glucose-6-phosphatase activities of normal and STZ diabeticrats. The glucokinase activity was lower in the STZ-induceddiabetic rats compared with the control rats. The supplementationof genistein and ISP increased the glucokinase level of the STZ-induced diabetic rats. The glucose-6-phosphatase level was in-creased in diabetic rats. A significant reduction in glucose-6-phosphatasewas observed in the groups treatedwith genistein andISP compared with the diabetic control group. The effects weremore potent in the STZ-ISP group than in the STZ-G group.

Serum and hepatic lipids

The total cholesterol and triglyceride concentrations in theserum and liver were significantly higher in the STZ-induced

Table 3Effect of genistein and isolated soy protein on serum and hepatic lipids indiabetic rats


SerumTotal cholesterol(mmol/L)

2.40±0.16b 3.62±0.20a 2.61±0.09b 2.55±0.08b

HDL-cholesterol(mmol/L)

1.22±0.05a 0.42±0.05c 0.65±0.06b 0.63±0.06b

Triglyceride (mmol/L) 1.11±0.09b 2.03±0.16a 1.16±0.09b 1.15±0.13b

LiverCholesterol (mmol/g) 0.13±0.02b 0.23±0.04a 0.16±0.03b 0.15±0.03b

Triglyceride (mmol/g) 0.24±0.02b 0.45±0.05a 0.28±0.03b 0.26±0.03b

Values are mean±SD of 8 rats from each group.a,b,c Means in the same row not sharing a common superscript are significantlydifferent ( p<0.05) between groups.

diabetic rats than in the control rats (Table 3). The supplemen-tation of genistein and ISP suppressed the increase in the totalcholesterol and triglyceride levels in the serum and liver of thediabetic rats. The genistein and ISP supplementation decreased inthe serum and hepatic triglyceride levels significantly to almostthe control concentration. The HDL-cholesterol concentrationwas also significantly lowered by the induction of diabetes;however it was higher in the genistein and ISP supplementedgroups compared to the other diabetic group.

Antioxidant enzyme activities and TBARS concentration

The activities of SOD, CAT, GSH-Px and hepatic TBARSconcentration are given in Table 4. SOD and GSH-Px activitiesin the liver of the STZ-induced diabetic rats were significantlydecreased compared to the control rats. Administering genisteinand ISP to the STZ-induced diabetic rats significantly increasedthose enzyme activities. CATactivity of the rats treatedwith STZwas significantly decreased, while genistein and ISP supplementto the STZ-induced diabetic rats appeared to increase its activity.The effect was more pronounced in the ISP supplemented groupthan in the genistein-supplemented group.

The concentration of TBARS in the STZ-induced diabeticrats was significantly elevated, the genistein and ISP supplementdecreased it significantly to almost the control concentration.

Serum aminotransferase activity and plasma urea level

The activities of serum AST and ALT and plasma urea levelof control and experimental animals are given in Table 5.Supplement with the genistein and ISP along with STZ showed

Table 5Effect of genistein and isolated soy protein on serum ALTand ASTactivities andplasma urea level in diabetic rats


ALT (unit/mL) 35.55±1.62c 63.88±1.90a 42.09±1.06b 43.19±1.06b

AST (unit/mL) 70.60±2.13c 101.66±3.06a 82.19±1.56b 80.34±1.69b

Urea (mg/dL) 32.62±1.52c 61.60±2.52a 43.14±1.36b 41.06±2.02b

Values are mean±SD of 8 rats from each group.a,b,c Means in the same row not sharing a common superscript are significantlydifferent ( p<0.05) between groups.


significantly reduced levels of AST and ALT as compared withthose of the unsupplemented STZ-treated rats. The STZ-induceddiabetic group resulted in a significant increase in plasma ureaconcentration compared to the control group; Supplementationof genistein and ISP decreased the level of urea as comparedwith that of the STZ-treated group.

Discussion

Diabetes arises from destruction of the β-islet cells of thepancreas, due to degranulation or reduction of insulin secretion(Ramkumar et al., 2004). The elevation in plasma insulin in thegenistein and ISP-treated STZ-diabetic rats could be due to theinsulinotropic substances present in the fractions, which inducethe intact functional β-cells of the Langerhans islet to produceinsulin, or the protection of the functional β-cells from furtherdeterioration so that they remain active and produce insulin.Diabetic rats induced by STZ show an increased sensitivity tooxygen free radicals and hydrogen peroxide, the breakdownproducts of the liver, which impose oxidative stress in diabetesand would damage inner endothelial tissue; this would even-tually be directly responsible for high blood glucose (Reddi andBollineni, 2001). The experimentally induced diabetes signif-icantly (p<0.05) increased the fasting blood glucose level by148% of the control level. However, the treatment of STZ-induced diabetic rats with the genistein and ISP reduced theirblood glucose levels by 19.6% and 24.9%, respectively, incomparison to the diabetic group. The genistein and ISP sup-plementations improved glucose tolerance in diabetic rats. Itseems that the hypoglycemic effects of genistein and ISP aredue to the increased level of serum insulin and the enhancementof peripheral metabolism of glucose (Skim et al., 1999).Supplement with ISP was found to increase the plasma insulinlevel more effectively than genistein. It seems that ISP containsone or more constituents that could increase the bioavailabilityof genistein. Further study will be necessary to elucidate themechanism of blood glucose regulation by genistein and ISP.

HbAIC was found to increase in diabetic rats up to 176%.And this increase is directly proportional to the fasting bloodglucose levels (Saravanan and Pugalendi, 2005). Since thediabetic animals had prior higher blood glucose levels, elevatedlevels of HbAIC were observed. Genistein and ISP fed diabeticrats showed the decrease in HbAIC level, which seems to be thereduction of blood glucose level.

Insulin increases hepatic glycolysis by increasing the activityand amount of several key enzymes including glucokinase, phos-phofructokinase and pyruvate kinase. In the liver, glucokinase isan important regulator of glucose storage and disposal (Saravananand Pugalendi, 2005). In this study, glucokinase activity wasdecreased in the liver of diabetic fats which may be due to adeficiency of insulin.Genistein and ISP fed diabetic rat showed anelevated activity of glucokinase, which may be associated withreduced blood glucose. Therefore, the ISP appears to be morepotent than the genistein.

Insulin decreases gluconeogenesis by decreasing the activitiesof key enzymes such as glucose-6-phosphatase, fructose 1,6-bisphosphatase, phosphoenolpyruvate carboxykinase and pyru-

vate carboxykinase (Murray et al., 2000). Glucose-6-phosphataseplays an important role in glucose homeostasis in liver and kidney(Berg et al., 2002). In this study, the increased activity of glucose-6-phosphatase in liver of diabetic rats seems to be insulin de-ficiency. In genistein and ISP fed diabetic rats, the activity of thatenzyme was significantly reduced, which may be responsible forthe improved glycemic control. The ISP seems to be more potentthan the genistein, and contains other active constituents thatcould potentiate the effect.

Hyperglycemia also generates reactive oxygen species whichin turn cause lipid peroxidation and membrane damage in thisstudy (Hunt et al., 1988). In the current study, the concentrationof hepatic TBARS was significantly increased after treatment ofSTZ in the rats. The increased concentration of TBARS suggeststhat an increase in oxygen free radicals could be due to eithertheir increased production or decreased destruction (Kakkaret al., 1995). The level of hepatic TBARS in genistein and ISPsupplemented rats showed a significant reduction, which indi-cates a decreased rate of lipid peroxidation. Several studies haveshown increased lipid peroxidation in clinical and experimentaldiabetes (Sundaram et al., 1996; Kakkar et al., 1998). Thepresent results show increased lipid peroxidation in tissues of thediabetic group. The increase of oxygen free radicals in diabetescould be due to an increase of blood glucose level, since anautoxidation generates free radicals (Ivorra et al., 1989). STZ hasbeen shown to produce oxygen free radicals. Lipid peroxide-mediated tissue damages have been observed in the developmentof type I and type II mellitus. Previous studies have reported thatlipid peroxidation in liver, kidney, and brain of diabetic rats wasincreased (Latha and Pari, 2003; Venkateswaran and Pari, 2002).

Concerning to the changes in lipid peroxidation, the diabetictissue showed decreased activity of the key antioxidants SOD,CAT, glutathione, GPx, and glutathione S-transferase, which playan important role in scavenging the toxic intermediate of in-complete oxidation. The decrease in the activity of these anti-oxidants can lead to an excess availability of the superoxide anion(O2

−) and hydrogen peroxide in biological systems, which in turngenerate hydroxyl radicals resulting in initiation and propagationof lipid peroxidation. Administration of genistein and ISPincreased the activity of enzymes and may help to control freeradicals (Kumuhekar and Katyane, 1992).

The induction of SOD activity by genistein and ISP may beattributed to inhibition of the generation of active oxygen spe-cies from autoxidation of glucose generation from the action ofSTZ. The increased activity of SOD accelerates dismutation ofsuperoxide radicals to H2O2, which is removed by CAT. Thisindicates that the genistein and ISP supplements have altered theSOD, CAT, and GSH-Px activities and reduced oxidative stressin the diabetic rats, resulting in a lower TBARS concentration.

The diabetic hyperglycemia induces the elevation of plasmalevels of urea and creatinine which are considered as sig-nificant markers of renal dysfunction (Almdal and Vilstrup,1988). In this study plasma urea in the diabetic groups in-creased by 88.8% of the control level. While after the supple-ment of genistein and ISP to the diabetic rats, the level of ureawas significantly ( p<0.05) decreased in plasma by 30.0% and32.5%, respectively, compared with the diabetic group. This


result indicates that genistein and ISP are capable of amelio-rating the impaired diabetic kidney function in addition to itshypoglycemic control.

Increased activities of AST and ALT are used as indices ofliver damage.When the liver is damaged, these enzymes leak outof liver cells in large quantities, so their concentrations in theblood are increased (Tawta et al., 2000). Possible explanation forthe differential effects of genistein and ISP on the activities ofASTand ALT in plasma is that genistein and ISP may inhibit theliver damage induced by STZ.

Genistein and ISP would appear to contribute to alleviatingthe adverse effect of diabetes mellitus by enhancing the lipidmetabolism as well as the hepatic antioxidant defense system.Genistein and ISP supplements may be beneficial for correctingthe hyperglycemia and preventing diabetic complications. TheISP appears to be more potent than the genistein. Hence furtherbiochemical and pharmacological studies are being carried outto elucidate their mechanism of action.

Acknowledgements

The author is grateful for the financial support provided bythe foundation of Kosin University.

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