LFS-14051; No of Pages 7 Life Sciences xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Life Sciences journal homepage: www.elsevier.com/locate/lifescie

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Md. Maroof Alam 1, Dilnasheen Meerza, Imrana Naseem

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Department of Biochemistry, Faculty of Life Sciences, AMU, Aligarh 202002, India

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Article history: Received 14 February 2014 Accepted 6 June 2014 Available online xxxx

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Keywords: Quercetin Hyperglycemia Type 2 diabetes GLUT4 DNA damage

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Protective effect of quercetin on hyperglycemia, oxidative stress and DNA damage in alloxan induced type 2 diabetic mice

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Aims: Quercetin is a natural polyphenolic flavonoid and acts as a quencher for reactive oxygen species generated by any physical or chemical action. In type 2 diabetes mellitus (T2DM) the basic characteristic feature is hyperglycemia which leads to complications involving oxidative stress. In view of this, the present study was conducted to examine the effect of quercetin in T2DM. Main methods: A total of 18 mice were divided into three groups, vis control, diabetic and diabetic treated with quercetin. Fasting blood glucose (FBG) levels and anti-oxidant enzyme activity were assayed. Creatinine, urea, lipid peroxidation, GLUT4 expression and DNA damage were also measured. Key findings: A significant decrease in FBG level and liver and kidney marker enzymes was observed in the quercetin treated group as compared to the diabetic one. Glutathione, SOD, catalase, and glutathione-S-transferase levels were also found to be increased on quercetin supplementation. Thiobarbituric acid-reactive substance level was decreased while GLUT4 expression levels were increased in the treated group. DNA damage was also affected positively by quercetin when subjected with single cell alkaline gel electrophoresis. Thus, we may suggest an anti-oxidant potential and protective effect of quercetin in T2DM mice. Significance: From this study, we conclude that quercetin ameliorates hyperglycemia and oxidative stress, by blunting free radical induced toxicity in T2DM. © 2014 Published by Elsevier Inc.

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Introduction

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Type 2 diabetes, is the fastest growing epidemic of the 21st century, accounting for 90% of the cases globally. The number of diabetics is expected to rise from 366 million in 2011 to 552 million in 2030 (Narayan et al., 2006). According to pathophysiological database, individuals with type 2 diabetes have three cardinal abnormalities: (a) resistance to the action of insulin in peripheral tissues; (b) defective insulin secretion especially in response to glucose; and (c) increased production of glucose by the liver. Such abnormalities may lead to the precipitation of type 2 diabetes mellitus (T2DM). In T2DM the basic characteristic feature is hyperglycemia (increased glucose level) which leads to complications involving oxidative stress. The age-associated decline in antioxidant defense system results in oxidative stress (Ames et al., 1993; Paolisso and Giugliano, 1996), which could then adversely affect glucose metabolism (Paolisso and Giugliano, 1996). Oxygen-derived radicals like superoxide anion and hydrogen peroxide have been shown to

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E-mail address: [email protected] (M.M. Alam). Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, U.P. 202002, India. Tel.: +91 571 2700741 [O], +91 9719069125 [R]; fax: +91 571 2706002 [O]. 1

inactivate key enzymes of glucose metabolism in both the glycolytic pathway and the electron transport chain coupled to oxidative phosphorylation (Janero et al., 1994). Further oxidative stress also causes depolarization and calcium uptake in insulin producing cells (Wahl et al., 1998), a phenomenon that stimulates insulin secretion, which could potentially result in hyperinsulinemia. Oxidative stress has been implicated in the development of chronic diabetic complications (Oberley, 1988). Oxidative stress can damage several biomolecules like proteins, lipids and DNA, thereby leading to the inactivation of enzymes affecting DNA integrity and cellular membrane composition. Studies have shown that compounds with strong antioxidant property can potentially be effective in delaying diabetes related complications. One such class of compounds is plant-derived polyphenols, which can be divided into several groups like flavonols (e.g. quercetin, kaempferol, catechin), flavones (e.g. luteolin) and isoflavones (e.g. genistein) (Narayan et al., 2006; Tapas et al., 2008). For this study we chose quercetin which belongs to flavonols. Quercetin (QC) is the most abundant of all flavonoids and is found in green vegetables, onions, apples, legumes, green tea, citrus fruits, red grape wine, etc. (Baghel et al., 2012). Quercetin, like other flavonoids, acts as a quencher for reactive oxygen species generated by any physical or chemical action (Saija et al., 1995). Moreover, quercetin inhibits xanthine oxidase and lipid peroxidation as well as cyclooxygenase and

http://dx.doi.org/10.1016/j.lfs.2014.06.005 0024-3205/© 2014 Published by Elsevier Inc.

Please cite this article as: Alam MM, et al, Protective effect of quercetin on hyperglycemia, oxidative stress and DNA damage in alloxan induced type 2 diabetic mice, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.06.005

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Materials and methods

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Study population

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The study was conducted on Swiss albino mice. Mice were randomly divided into three groups of 6 each. The first group (C) was kept untreated as control and was given saline. The second (D) and the third groups (Q) were subjected to a single intra-peritoneal injection of alloxan (150 mg/kg body weight) and their fasting blood glucose (FBG) was monitored till they became diabetic. Third group (Q) diabetic induced mice were orally supplemented with quercetin (20 mg/kg body weight) for a regular period of three weeks.

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Sample collection

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Overnight fasting mice were sacrificed by cervical dislocation at the end of the 21 day treatment period and their blood and tissue samples were collected for analysis. 2–3 ml blood was collected in sterilized centrifuge tubes which were immediately centrifuged at 1000 ×g for 15 min. Furthermore, the serum parameters were analyzed on the same day while the tissues (liver, kidney, pancreas and skeletal muscle) were kept in Hepes's buffer at −20 °C for further enzyme analysis.

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Biochemical analysis

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Fasting blood glucose level, the activities of glucose metabolic enzymes (hexokinase, FBPase and G6Pase), anti-oxidant enzymes like Cu–Zn superoxide dismutase (CuZn SOD), and catalase were assayed with standard protocols along with levels of cellular reductants (GST and SH) and MDA levels. Liver and kidney function tests were performed. GLUT-4 expression and DNA damage were also estimated in all the groups.

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Fasting blood glucose estimation

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Glucose levels were estimated by the glucose oxidase–peroxidase method (Trinder, 1969) using a Ranbaxy diagnostic kit. Briefly 10 μl of the fasting blood samples was mixed with 1 ml of working solution (containing 6.7 U/ml of glucose oxidase, 6.2 U/ml of horseradish peroxidase, 0.2 Mm of 4-aminoantipyrine, 8 M of phosphate buffer and 86 mM of phenol). The blank and standard solutions were also prepared simultaneously by adding 10 μl of distilled water and 10 μl of standard glucose (100 mg/dl) to 1 ml of working solution. All the tubes were mixed well and incubated at room temperature for 30 min and absorbance was taken at 505 nm.

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Estimation of glucose metabolic enzymes

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Hexokinase activity The hexokinase activity was measured by the method of Crane and Sols (1953). The tissues were collected and homogenized in sodium phosphate buffer. The reactions were carried out at 37 °C by adding 1 ml of reaction mixture containing Tris–HCl (50 μM), MgCl2 (10 μM),

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Fructose-bisphosphatase activity (FBPase) The FBPase activity was measured by the method of Freedland and Harper (1959). The reaction was carried out by adding 1.5 ml of reaction mixture containing Tris–HCl (50 μm), MgCl2 (10 μm), cysteine HCl (12 μm), and fructose 1,6 bisphosphate (10 μm) to the sample containing 0.6–0.8 mg of protein. The reaction was stopped by adding 1 ml of 10% TCA after 60 min and the samples were then centrifuged at 2000 ×g for 10 min. The phosphate released was estimated by the method of Tausky and Shorr (1953) in the supernatant.

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Glucose-6-phosphatase (G6Pase) activity G6Pase activity was assayed according to the method of Shull et al. (1956). The reaction mixture in a total volume of 1.5 ml contained Tris–HCl buffer (50 μm); MgCl2 (10 μm); glucose-6-phosphate (10 μm); and sample. The reaction was carried out at 37 °C and stopped with 1 ml of 10% TCA after 60 min. The samples were centrifuged at 2000 ×g for 10 min and phosphorous was estimated in the protein free supernatant by the method of Tausky and Shorr (1953).

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Estimation of antioxidant enzymes

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Superoxide dismutase (CuZn SOD) The assay of SOD was conducted according to the method of Marklund and Marklund (1947) on the basis of autoxidation of pyrogallol. In this procedure, 2.85 ml of Tris–succinate buffer (0.05 M, pH 8.2) was added to 50 μl of sample followed by 100 μl of pyrogallol (8 mM) under dark condition. The change in absorbance was read at 412 nm on a Centra-5 spectrophotometer for 3 min. References were also taken for every 10 samples. The specific activity was reported in units per mg of protein.

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Catalase (CAT) The activity of catalase was measured by the protocol of Aebi (1984). The reaction mixture containing 1.95 ml of potassium phosphate buffer (50 mM, pH 7.0), 50 μl of the sample and 1 ml of hydrogen peroxide (30 mM) was incubated under dark condition. The absorbance was immediately recorded at 240 nm for 3 min. Catalase activity was calculated as nmol of H2O2 consumed per mg of protein per minute.

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Glutathione reduced (GSH) For the estimation of total GSH level in the samples, the Jollow et al. (1974) method was implemented. According to this protocol, 0.5 ml of sample homogenate was mixed with equal volume of 4% (w/v) sulfosalicylic acid and incubated at 4 °C for 1 h followed by centrifugation at 1200 g for 15 min. The supernatant (0.2 ml) was carefully taken out and was mixed with 1.1 ml of potassium phosphate (0.1 M, pH 7.4). The reaction was initiated by the addition of 0.2 ml of DTNB which was read at 412 nm within 30 s. The GSH level was reported in nmol per gram of the tissue.

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Estimation of cellular reductants

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Glutathione-S-transferase (GST) The assay of GST detoxifying enzyme was conducted by the protocol of Habig et al. (1974). The sample (0.1 ml) was mixed with 2.7 ml of GSH (1 mM) and 0.2 ml of CDNB (1 mM) in the sequence. After mixing well, it was read at 340 nm in the time span of 3 min against the blank containing all the reagents except the enzyme source (the sample). Its

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ATP (5 μM), and glucose (2 μM) to 1–1.5 mg of sample protein. The reaction was stopped after 1 h by adding 0.5 ml each of 10% Ba(OH)2 and ZnSO4 solutions. The samples were centrifuged at 2000 ×g for 10 min and glucose was estimated in supernatant (free from phosphorylated derivatives). The glucose estimation was determined by the method of Nelson (1994) with standard glucose solution ranging between 5 and 45 μg/ml.

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lipo-oxygenase which has been shown through in vitro and in vivo studies conducted on quercetin (Loggia et al., 1988; Kim et al., 1998). Loggia et al. have also shown that quercetin can protect pancreatic β cells from inflammatory damage. Another important function attributed to quercetin is the ability to reduce aldose reductase, an enzyme involved in the conversion of glucose to sorbitol via polyol pathway. It is the accumulation of sorbitol in different organs of the body that leads to various complications like diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, etc. (Lim et al., 2001; Goodarzi et al., 2006). Therefore, the present study was conducted to postulate the possible mechanism that can be affected by quercetin in maintaining redox status of the cell in T2DM mice.

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M.M. Alam et al. / Life Sciences xxx (2014) xxx–xxx

Please cite this article as: Alam MM, et al, Protective effect of quercetin on hyperglycemia, oxidative stress and DNA damage in alloxan induced type 2 diabetic mice, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.06.005

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Estimation of malondialdehyde (MDA) level

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MDA is the major stable product of lipid peroxidation. Its level was determined by the procedure of Beuge and Aust (1978). The reaction mixture was made with 0.5 ml homogenate with equal volume of TBA (0.67%) and TCA (30%) in centrifuge tubes which were incubated in a boiling water bath for 20 min. The tubes were later centrifuged at 4000 rpm for 15 min. The absorbance of the pink supernatant was read at 530 nm. The level was expressed in nmol of MDA formed per mg of the protein by using a molar extinction co-efficient of 1.56 × 10− 5 M− 1 cm− 1 for MDA–TBA colored complex.

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Estimation of renal function markers (RFTs) in serum

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Urea

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The level of urea in the serum samples was estimated by a commercially available kit (Span Diagnostics Limited, India) and was reported in mg per dl of the sample.

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Creatinine

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The level of creatinine in the serum samples was estimated by a commercially available kit (Span Diagnostics Limited, India) and was reported in mg per 100 ml of the sample.

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Alkaline phosphatase (ALP)

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The assay of ALP was carried out using a commercially available kit (Span Diagnostics Limited, India) based on Kind and King's (1954) method.The principle of the kit is that alkaline phosphatase converts phenyl phosphate to inorganic phosphate and phenol at pH 10. Phenol formed reacts in alkaline pH with 4-aminoantipyrine in the presence of the oxidizing agent potassium ferricyanide and results in the formation of an orange-red colored complex, which can be measured colorimetrically. The color intensity is proportional to the enzyme activity. The reaction can be represented as:

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alkaline phosphatase ðpH 10:0Þ

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phenol þ 4‐aminoantipyrine

potassium ferricyanide

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phenol þ phosphate 248 249

orange‐red complex: 251

GLUT-4 estimation

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Sulfahydryl groups (\SH) The estimation of\SH level was conducted by the method of Sedalk and Lindsay (1968). For this, 100 μl of the sample was added to 2.5 ml of Tris–HCl (0.1 M, pH 8), 0.3 ml of EDTA and 0.1 ml of DTNB. The reaction mixture was vortexed and kept at room temperature for 30 min which was then read at 412 nm. Its level was expressed in nmol per gram of tissues.

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with the help of the standard curve provided with the kit and was 235 expressed in units per ml. 236

specific activity was calculated taking a millimolar extinction coefficient value of 9.6 mM−1 cm−1 of CDNB and was expressed in nmol of CDNB conjugates formed per mg of the protein.

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Glutamate pyruvate transaminase (GPT) or alanine transaminase (ALT)

Comet assay (single cell alkaline gel electrophoresis)

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GPT activity was assayed by a commercial kit (Span Diagnostics Limited, India) that was based on the protocol of Reitman and Frankel (1957). The enzyme catalyzes the following reaction

Comet assay was performed in an alkaline condition in accordance with the protocol of Singh et al. (1988) with minor modifications. Fully frosted slides were precoated with 1% normal melting agarose. Cells isolated from each organ cell suspension were mixed with 80 μl 1% low melting point agarose to form the working cell suspension separately for each organ. This suspension was pipetted over the first layer and covered with coverslips. The slides were placed on ice packs for 8–10 min to solidify the agarose. The coverslips were gently removed and a third layer of 0.5% low melting point agarose was pipetted. The slides were covered with coverslips and were placed on ice packs to solidify. The coverslips were then removed and the slides were immersed in ice cold lysis buffer for 2–3 h. After lysis, the DNA was allowed to unwind in alkaline electrophoretic solution (300 mM NaOH, 1 mM EDTA, pH ≤ 13). Electrophoresis was performed at 4 °C, in a field strength of 0.7 V/cm and 330 mA current. The slides were neutralized with ice cold 400 mM Tris (pH 7.5), and stained with 75 μl EtBr and covered with coverslips. Slides were scored using an image analysis system (Komet 5.5, Kinetic Imaging, Liverpool, UK) attached to an Olympus (CX41) fluorescent microscope (Olympus Optical Co., Tokyo, Japan) and a COHU 4910 integrated CC camera (equipped with 510–560 nm excitation and 590 nm barrier filters) (COHU, San Diego, CA, USA). Images of 25 cells were analyzed from each triplicate slide. Tail length (migration of DNA from nucleus in μm) was the parameter used to asses DNA damage.

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GPT

α‐ketoglutarate þ L‐alanine → L‐glutamate þ pyruvate:

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The method involves the reaction with pyruvate formed with DNPH to produce brown colored hydrazones which is read at 505 nm taking distilled water as blank. The activity was reported in units per ml of the sample by the standard plot provided with the kit.

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Glutamate oxaloacetate transaminase (GOT) or aspartate transaminase (AST) The assay of GOT was also carried out by a commercially available kit (Span Diagnostics Limited, India) based on the Reitman and Frankel (1957) method. The enzyme catalyzes the reaction given below: GOT

α‐ketoglutarate þ L‐aspartate → L‐glutamate þ oxaloacetate: 233 234

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Estimation of liver function markers (LFTs) in serum

Quantitative determination of GLUT-4 was done in serum and tissue homogenates (adipocytes and skeletal muscles) using an ELISA kit following manufacturer's instructions. The microtiter plate was precoated with an antibody specific to GLUT-4. Samples were added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for GLUT-4, followed by the addition of avidin conjugated to horseradish peroxidase (HRP) to each microplate well and incubated, followed by the addition of TMB substrate solution. Only in those wells where GLUT-4 was bound with a biotin-conjugated antibody and enzyme-conjugated avidin did the enzyme–substrate reaction occur. The reaction was terminated by the addition of sulfuric acid solution and the color change was measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of GLUT-4 in the samples is determined by comparing the O.D. of the samples to the standard curve, supplied with the kit.

The product oxaloacetate reacts with DNPH to give brownish hydrazones that are observed at 505 nm. The activity was calculated

Please cite this article as: Alam MM, et al, Protective effect of quercetin on hyperglycemia, oxidative stress and DNA damage in alloxan induced type 2 diabetic mice, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.06.005

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M.M. Alam et al. / Life Sciences xxx (2014) xxx–xxx

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Statistical analysis

Effect of quercetin on lipid peroxidation

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All the data have been expressed in mean ± SEM for all continuous variables. Comparisons among various groups were conducted by one way-ANOVA with the help of software ‘Origin 6.1’ and ‘GraphPad Prism 5’. p ≤ 0.05 was chosen as statistically significant for the treatment. The experiments were repeated thrice to check the reproducibility of the results.

MDA levels have been widely used to determine lipid peroxidation in tissue and in cells in both clinical and experimental studies. Fig. 2 shows MDA levels; as assayed the values of the quercetin supplemented group were found to be close to the normal values while the highest levels were recorded for the diabetic group.

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Effect of quercetin on RFT and LFT in serum

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Previous studies have shown that T2DM leads to kidney and liver dysfunction. The levels of creatinine, urea, ALP, AST and ALT were found to be elevated in the diabetic group as depicted in Table 4. While the quercetin supplemented group showed significant recovery in all these parameters.

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Results

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Effect of quercetin on FBG level

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Fig. 1 shows the fasting blood glucose levels in all three groups. The FBG was found to be increased in the diabetic group (250 ± 0.41 mg/dl), confirming the establishment of diabetes compared to the control group (118 ± 0.21 mg/dl). The diabetic group supplemented with quercetin showed a significant decrease in the FBG levels (160 ± 0.37 mg/dl) suggesting its ameliorating role in the pathogenesis of hyperglycemia.

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Effect of quercetin on the activities of glucose metabolic enzymes

Role of quercetin on DNA damage in T2DM

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Table 1 shows the changes in the activities of all three enzymes. Hexokinase activity was found to be decreased in the diabetic group (liver, kidney and skeletal muscle) which recovered upon quercetin supplementation, while FBPase and G6Pase activities decreased with quercetin treatment.

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Effect of quercetin on the activities of antioxidant enzymes

Generation of superoxide and hydroxyl radicals may lead to DNA damage in T2DM. This was tested using the comet assay. The average tail length of the comet, a measure of DNA damage was found to be significantly higher in the diabetic group compared to control (Table 5 and Fig. 4A and B). However, quercetin supplementation showed a significant recovery in DNA damage as evident from the decreased average tail length of the comet.

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Tables 2 and 3 show enzyme activity of the antioxidant enzyme SOD, catalase, GST and sulfahydryl as assayed in the samples. Antioxidant enzyme activity was found to be the highest in the control group. The specific activity of all the enzymes decreased significantly in the diabetic group. The enzymes showed a recovery of activity in the group supplemented with quercetin. SOD was the most affected among all the enzymes with a 26% and 23% decline in liver and kidney activities respectively. GST and catalase were also affected exhibiting a significant decrease in the activity in the liver and kidney of the diabetic animal. SOD, GST and catalase showed a significant recovery in the liver, kidney, pancreas and skeletal muscles upon quercetin supplementation. The total sulfahydryl group was found to be elevated in the diabetic group and was recovered in the quercetin treated group.

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Role of quercetin in GLUT-4 expression level

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Fig. 1. Fasting blood glucose level. Fasting blood glucose levels in control, diabetic and quercetin (20 mg/kg bw) treated mice. All the data have been expressed in mean ± SEM for five different preparations of each sample of three independent experiments. * indicates significantly different from control at p ≤ 0.05. # indicates significantly different from the diabetic group at p ≤ 0.03.

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Fig. 3 shows a decrease in GLUT-4 expression levels in the adipo- 341 cytes, skeletal muscle and serum of the diabetic group. While quercetin 342 supplementation increases the GLUT4 expression levels. 343

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Discussion

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Quercetin, a polyphenol of the flavonol group is found in abundance in green vegetables, green tea, citrus fruits, and red grape wine. Its protective role has been reported in animal models of type 1 diabetes. In the present study we have shown that quercetin works as an anti-diabetic agent via targeting both hyperglycemia and oxidative stress. This is shown by the reduction in fasting blood glucose levels in the quercetin supplemented group. Hyperglycemia, if not treated may lead to (a) β-cell glucotoxicity, (b) increased flux through the polyol pathway and (c) increased production of advanced glycation end products (AGEs) (Rolo and Palmeira, 2006). Quercetin was reported to possess α-glucosidase inhibitory activity in vitro (Ishikawa et al., 2007; Jo et al., 2009). The α-glucosidase inhibitors act as competitive inhibitors of the enzymes needed to digest carbohydrates. Inhibition of this enzyme reduces the rate of digestion of carbohydrates. The acute consumption of quercetin was found to be effective in controlling postprandial blood glucose in streptozotocin or alloxan induced diabetic animals (Kim et al., 2011). Estimation of enzyme activities of hexokinase/glucokinase, glucose-6phosphatase and fructose-bisphosphatase showed a distinct pattern. The decreased hexokinase activity in the diabetic group is an evidence of the fact that there is an insufficient amount of glucose reaching the cell even in the state of hyperglycemia, whereas increased activities of G6Pase and FBPase suggest an increase in the gluconeogenic pathway which contributes to increased glucose production. However, the pattern of activities of these enzymes was found to be reversed in the quercetin supplemented group, showing an increase in hexokinase and a decrease in G6Pase and FBPase activities, thereby providing an insight on the effect of quercetin on glucose metabolism which eventually leads to the maintenance of glucose homeostasis and control of hyperglycemia. Oxidative stress also plays a crucial role in the development of diabetic complications (Wolff, 1993). In the pancreas the oxidative defense is already weak. In a diabetic state, the glucose auto-oxidation leads to the formation of more free radicals (Mullarkey et al., 1990). This further

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Please cite this article as: Alam MM, et al, Protective effect of quercetin on hyperglycemia, oxidative stress and DNA damage in alloxan induced type 2 diabetic mice, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.06.005

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M.M. Alam et al. / Life Sciences xxx (2014) xxx–xxx

t1:5

Parameters

Samples

Control

t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14

Hexokinase (μg/mg/prt)

Liver Kidney Skeletal muscle Liver Kidney Skeletal muscle Liver Kidney Skeletal muscle

2.03 0.36 1.44 0.63 0.90 1.11 1.27 1.72 2.43

1.94 0.31 1.40 0.70 1.21 1.28 1.39 1.81 2.53

± ± ± ± ± ± ± ± ±

0.09* 0.01* 0.01* 0.04* 0.08* 0.07* 0.03* 0.07* 0.05*

± ± ± ± ± ± ± ± ±

0.08# * 0.01# * 0.01# * 0.02# * 0.05# * 0.09# * 0.04# * 0.03# * 0.02# *

Table 2 Effect of quercetin on the activities of antioxidant enzymes in alloxan induced diabetic mice. SOD, catalase and GSH were measured in control, diabetic and quercetin supplemented groups. Target organs were the liver, kidney, pancreas and skeletal muscle. All the data have been expressed in mean ± SEM for five different preparations of each sample of three independent experiments. * indicates significantly different from control at p ≤ 0.05. # indicates significantly different from the diabetic group at p ≤ 0.05. Parameters

Sample

Control

t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17

SOD (unit/mg/prt)

Liver Kidney Pancreas Skeletal muscle Liver Kidney Pancreas Skeletal muscle Liver Kidney Pancreas Skeletal muscle

183.13 30.50 45.01 68.19 43.17 39.53 59.93 40.12 24.68 35.56 34.88 198.45

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Glutathione reduced (GSH) (unit/mg/prt)

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Catalase (unit/mg/prt)

Diabetic

± ± ± ± ± ± ± ± ± ± ± ±

9.2 1.76 2.11 2.20 1.98 1.43 1.39 1.55 0.98 0.96 1.08 5.3

D

t2:5

385 386

1.30 0.19 0.62 0.94 1.96 1.79 1.89 2.60 3.44

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G6Pase (μg/mg/prt)

0.10 0.01 0.03 0.02 0.03 0.04 0.06 0.03 0.08

Quercetin supplemented

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t2:1 t2:2 t2:3 t2:4

FBPase (μg/mg/prt)

± ± ± ± ± ± ± ± ±

Diabetic

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t1:3 t1:4

Table 1 Effect of quercetin on glucose metabolic enzymes in alloxan induced diabetic mice. Hexokinase, FBPase and G6Pase activities were measured in control, diabetic and quercetin supplemented groups. All the data have been expressed in mean ± SEM for five different preparations of each sample of three independent experiments. * indicates significantly different from control at p ≤ 0.05. # indicates significantly different from the diabetic group at p ≤ 0.05.

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t1:1

Q3 t1:2

5

137.42 21.07 20.21 31.09 25.62 22.74 30.62 26.95 16.43 22.24 19.76 168.89

± ± ± ± ± ± ± ± ± ± ± ±

Quercetin supplemented 7.2* 1.75* 1.22* 1.76* 1.11* 0.98* 1.09* 0.89* 1.09* 1.13* 1.03* 6.28*

170.86 26.32 33.72 59.45 40.85 35.18 52.71 36.98 22.92 32.11 31.65 181.80

± ± ± ± ± ± ± ± ± ± ± ±

9.7# * 1.56# * 1.33# * 2.78# * 1.34# * 1.12# * 1.78# * 1.24# * 0.91# * 0.89# * 0.97# * 4.8# *

t3:1 t3:2 t3:3 t3:4

Table 3 Effect of quercetin on the activities of GST and sulfahydryl groups in alloxan induced diabetic mice. GST and sulfahydryl groups were measured in control, diabetic and quercetin supplemented groups. Target organs were the liver, kidney, pancreas and skeletal muscle. All the data have been expressed in mean ± SEM for five different preparations of each sample of three independent experiments. * indicates significantly different from control at p ≤ 0.05. # indicates significantly different from the diabetic group at p ≤ 0.05.

394 395 396 397 398 399 400 401

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R

392 393

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390 391

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the organ damage. This will lead to an increase in the markers like alkaline phosphatase, aspartate transaminase, alanine transaminase, serum creatinine and urea. These markers were estimated and were found to be significantly improved upon quercetin supplementation. It is well known that alloxan causes diabetes by the reduction of alloxan to dialuric acid by a redox cycle with the formation of superoxide anion. These anions undergo dismutation to H2O2. Thereafter, highly reactive hydroxyl radicals can be formed if a divalent metal like Cu(II) is present by a Fenton like reaction. These ROS may cause selective damage of the pancreatic islet β-cells (Etuk, 2010). Based on our preliminary results, we propose that quercetin interacts with alloxan and blunts its oxidative potential. This significantly decreases the pancreatic β-cell damage and hence is able to slow down the process of development of diabetes when given in combination with alloxan. In addition the role of quercetin on L-type calcium channel (Bardy et al., 2013) as well as GLUT4 transporter is established. The next important target under investigation was the GLUT4 transporter. The expression and translocation of insulin-dependent GLUT4, which is predominantly

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402 Q9

enhances the oxidative stress leading to glucotoxicity. The major free radicals that play a role in T2DM are superoxide, hydroxyl and peroxyl radicals. These free radicals lead to DNA damage, protein modification, glycation reaction and oxidative modification of lipids (Hunt et al., 1990). Our results are in accordance with Baynes and Thorpe (1999) stating that diabetes mellitus increases oxidative stress in an animal model system. Moreover, confirmation about the recovery from oxidative stress in T2DM by quercetin was demonstrated through single cell alkaline gel electrophoresis. The average tail length in the QC supplemented group was found to be decreased almost three fold when compared with the diabetic group, thus illustrating the protective role of quercetin in DNA damage. The liver plays a critical role in maintaining blood glucose concentration through its ability to both supply glucose to the circulation via glycogenolysis and gluconeogenesis and, in the post-absorptive state, remove glucose from circulation after meal ingestion. The kidney is also equally affected as it is the chief excretory organ of the system and hyperglycemia forces the kidney to reabsorb excess glucose. This affects the liver and kidney leading to the impairment in

t3:5

Parameters

Sample

Control

t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13

GST (unit/mg/prt)

Liver Kidney Pancreas Skeletal muscle Liver Kidney Pancreas Skeletal muscle

112.86 66.31 33.67 78.01 0.254 0.351 0.96 1.12

Sulfahydryl group (μmol/mg/prt)

Diabetic ± ± ± ± ± ± ± ±

4.13 2.0 1.29 2.44 0.01 0.01 0.04 0.06

69.20 23.27 20.01 52.11 0.581 0.825 1.56 1.43

± ± ± ± ± ± ± ±

Quercetin supplemented 2.13* 1.11* 0.96* 1.98* 0.03* 0.04* 0.08* 0.09*

88.74 46.58 28.91 71.0 0.278 0.37 1.21 1.31

± ± ± ± ± ± ± ±

3.11# * 1.88# * 1.08# * 2.08# * 0.03# * 0.02# * 0.08# * 0.05# *

Please cite this article as: Alam MM, et al, Protective effect of quercetin on hyperglycemia, oxidative stress and DNA damage in alloxan induced type 2 diabetic mice, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.06.005

403 404 405 406 Q10 407 408 409 410 411 412 413 414 415 416 417 418 419 420

6

M.M. Alam et al. / Life Sciences xxx (2014) xxx–xxx Table 5 Average tail length of comet in μm of the pancreas, kidney and liver in alloxan induced diabetic mice. Average tail length of DNA damage in the case of control, diabetic and quercetin treated groups. Target organs were the liver, kidney and pancreas. All the data have been expressed in mean ± SEM for five different preparations of each sample of three independent experiments. * indicates significantly different from control at p ≤ 0.05. # indicates significantly different from the diabetic group at p ≤ 0.01.

Pancreas Kidney Liver

Control

Diabetic

Quercetin supplemented

t5:8

5.25 ± 0.21 6.5 ± 0.28 5.75 ± 0.15

35.25 ± 1.5* 32.0 ± 1.20* 30.5 ± 1.10*

13.75 ± 0.80# * 11.5 ± 0.72# * 12.5 ± 0.88# *

t5:9 t5:10 t5:11

Parameters

Control

t4:8 t4:9 t4:10 t4:11 t4:12

Creatinine (mg/100 ml) Urea (mg/dl) ALP (KA units) AST (U/l) ALT (U/l)

0.52 38.5 7.85 25.26 32.02

Diabetic 0.01 0.21 0.11 0.45 0.51

0.72 53.33 45.16 33.3 41.20

± ± ± ± ±

R O

Quercetin supplemented 0.01* 0.42* 0.43* 0.71* 0.45*

0.55 42.30 12.13 26.50 33.72

± ± ± ± ±

0.01# * 0.55# * 0.28# * 0.34# * 0.82# *

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N

C

O

R

± ± ± ± ±

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t4:7

C

T

Table 4 Effect of quercetin on the parameters of renal function test and liver function test in serum. Mentioned parameters were recorded in control, diabetic and quercetin supplemented groups. All the data have been expressed in mean ± SEM for five different preparations of each sample of three independent experiments. * indicates significantly different from control at p ≤ 0.05. # indicates significantly different from the diabetic group at p ≤ 0.05.

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t4:1 t4:2 t4:3 t4:4 t4:5 t4:6

P

expressed in adipocytes and skeletal muscle cells are also increased. 422 This functionally regulates glucose uptake into adipose tissues in re423 sponse to elevated levels of insulin in circulation (Huang and Czech, 424 2007; Leto and Saltiel, 2012). In response to insulin, intracellular vesicle 425 containing glucose transporter proteins translocates to the cell surface 426 Q11 and thus glucose uptake is increased (Brannmark et al., 2013; Tatyana

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and Konstantin, 1999). The enhancement of GLUT4 level is responsible for the increment in glucose uptake and antidiabetic property of quercetin. Quercetin supplementation reversed hyperglycemiainduced decline of the Akt phosphorylation level. It is assumed that quercetin may activate insulin receptor substrates which leads to the initiation of the PI3K/AKT pathway by phosphorylation (Pessin and Salteil, 2000). This is subsequently needed for the metabolic effect of insulin and is responsible for the activation of glycogen synthase for the observed antidiabetic effect of quercetin. In our finding the quercetin supplemented group showed a significant increase in GLUT4 expression level. These results demonstrate that quercetin enhances glucose uptake and ameliorates the hyperglycemic effect via GLUT4 expression also. Thus, quercetin supplementation potentially can lower the complications by controlling hyperglycemia and oxidative stress parameters and by inducing expression of GLUT4 via mRNA expression and translocation to the plasma membrane (Jing et al., 2014). This may be very useful in the management of T2DM.

E

Fig. 2. Lipid peroxidation. MDA levels in the liver, kidney and pancreas of control, diabetic and quercetin (20 mg/kg bw) treated mice. All the data have been expressed in mean ± SEM for five different preparations of each sample of three independent experiments. * indicates significantly different from control at p ≤ 0.05. # indicates significantly different from the diabetic group at p ≤ 0.03.

Q2

Fig. 3. GLUT-4 expression level. GLUT-4 expression levels in the adipocytes, skeletal muscle and pancreas in control, diabetic and quercetin (20 mg/kg bw) treated mice. All the data have been expressed in mean ± SEM for five different preparations of each sample of three independent experiments. * indicates significantly different from control at p ≤ 0.05. # indicates significantly different from the diabetic group at p ≤ 0.05.

t5:1 t5:2 t5:3 t5:4 t5:5 t5:6 t5:7

Fig. 4. Comet assay. (A) The images of single cell gel electrophoresis. (B) Average tail length of DNA damage in the case of control, diabetic and quercetin supplemented groups. Target organs are the liver, kidney and pancreas. All the data have been expressed in mean ± SEM for five different preparations of each sample of three independent experiments. * indicates significantly different from control at p ≤ 0.05. # indicates significantly different from the diabetic group at p ≤ 0.01.

Please cite this article as: Alam MM, et al, Protective effect of quercetin on hyperglycemia, oxidative stress and DNA damage in alloxan induced type 2 diabetic mice, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.06.005

427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443

M.M. Alam et al. / Life Sciences xxx (2014) xxx–xxx

444

Conclusion

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450 451

In the present study, we suggest that supplementation with dietary antioxidants especially quercetin might help to reduce damage brought about by free radical toxicity in T2DM. The most important aspect of this flavonoid is its availability and cost, as it is found in a wide variety of fruits and vegetables and therefore can be included in the human diet. However more studies are required to get a deep insight into the mode action of quercetin.

452

Conflict of interest statement

447 448 449

453

The authors declare that there are no conflicts of interest.

456

Acknowledgments

457

O

Chen et al., 1990

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455

The authors acknowledge the financial assistance provided by the

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Aligarh Muslim University. We are also thankful to Dr. Sandesh Chibbar, Dr. Iftekhar Hassan, all the friends, lab colleagues and fellows who directly or indirectly helped us during different phases of treatment and experimentation in this work.

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458 Q13 University Grant Commission (UGC), New Delhi under SAP program, 459 DST-FIST and the facilities provided by the Department of Biochemistry, 460 461

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Please cite this article as: Alam MM, et al, Protective effect of quercetin on hyperglycemia, oxidative stress and DNA damage in alloxan induced type 2 diabetic mice, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.06.005

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