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Influence of rutin treatment on biochemical alterations in experimental diabetes
Biomedicine & Pharmacotherapy 64 (2010) 214–219
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Influence of rutin treatment on biochemical alterations in experimental diabetes Ana Ange´lica Henrique Fernandes a,*, Ethel Lourenzi Barbosa Novelli a, Katashi Okoshi b, Marina Politi Okoshi b, Bruno Paulino Di Muzio a, Julliano F. Campos Guimara˜es b, Ary Fernandes Junior c a b c
Department of Chemistry and Biochemistry, Institute of Biosciences, Sa˜o Paulo State University, UNESP, Botucatu, CEP 18618-000, Sa˜o Paulo, Brazil Department of Clinical and Cardiology, Faculty of Medicine, Sa˜o Paulo State University, UNESP, Botucatu, Sa˜o Paulo, Brazil Department of Microbiology and Immunology, Institute of Biosciences, Sa˜o Paulo State University, UNESP, Botucatu, Sa˜o Paulo, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 June 2009 Accepted 24 August 2009 Available online 27 October 2009
Dietary antioxidant compounds such as flavonoids may offer some protection against early-stage diabetes mellitus and its complications. Abnormalities in both glucose metabolism and lipid profile constitute one of the most common complications in diabetes mellitus. The present study aimed to evaluate the effect of rutin, through biochemical parameters, on experimental streptozotocin (STZ)induced diabetes in rats. Male Wistar rats were divided into four groups: untreated controls (GI); normal rats receiving rutin (GII); untreated diabetics (GIII); diabetic rats receiving rutin (GIV). STZ was injected at a single dose of 60 mg kg-1 to induce diabetes mellitus. The diabetes resulted in increased serum glucose, cholesterol, triacylglycerols and lipoproteins (LDL and VLDL-cholesterol) but decresed serum HDL-cholesterol and insulin. Rutin (50 mg kg–1) reduced (p < 0.05) blood glucose and improved the lipid profile in STZ-induced diabetic rats. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activities were significantly augmented in serum of STZ-diabetic rats, while these activities were diminished in hepatic and cardiac tissues compared with the control group. Rutin prevents changes in the activities of ALT, AST and LDH in the serum, liver and heart, indicating the protective effect of rutin against the hepatic and cardiac toxicity caused by STZ. Rutin was associated with markedly decreased hepatic and cardiac levels of tryacylglycerols and elevated glycogen. These results suggest that rutin can improve hyperglycemia and dyslipidemia while inhibiting the progression of liver and heart dysfunction in STZ-induced diabetic rats. ß 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Diabetes Dyslipidemia Rutin
1. Introduction Diabetes mellitus is a heterogeneous metabolic disorder characterized by hyperglycemia resulting from defective insulin secretion, resistance to insulin action or both [1,2] and in stimulating glucose uptake in tissues. Type 1 diabetes is the consequence of an autoimmune-mediated destruction of pancreatic b-cells, leading to insulin deficiency [3]. The disease is characterized by disturbance of carbohydrate and lipid metabolism, and is diagnosed by the presence of hyperglycemia [4]. The clinical manifestation of diabetes is associated with the development of certain diabetic complications [5]. In patients with diabetes, the abnormalities in lipid metabolism generally lead to elevated levels of serum lipids and lipoproteins that, in turn, play an important role in the occurrence of premature and severe atherosclerosis [6,7]. Furthermore, type 1 diabetes leads to dyslipidemia with elevated levels of total and low density
* Corresponding author. Tel.: +55 14 38116255; fax: +55 14 38116255. E-mail address: [email protected] (A.A.H. Fernandes). 0753-3322/$ – see front matter ß 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2009.08.007
lipoprotein (LDL) cholesterol, elevated tryacylglicerols and low levels of high density lipoprotein (HDL) cholesterol [8,9]. Since streptozotocin (STZ) has been recognized as toxic to bcells of the Islets of Langerhans, it has been widely utilized to induce type 1 diabetes mellitus with concomitant insulin deficiency [10]. STZ-induced diabetes in rats is one of the animal models of human diabetes mellitus [11]. After insulin treatment became available, evidence emerged suggesting that human diabetes mellitus has a multifactorial etiology. Insulin was and still is the principle hypoglycemic medication used in diabetes mellitus treatment. In order to discover other hypoglycemic agents, many investigations have been performed on traditional medicines to test prospective hypoglycemic natural substances [12]. Natural products used in folk medicine to treat diabetes represent a viable alternative for the control of this disease. Studies have examined the effects of natural flavonoids on physiological and pathological conditions of glucose metabolism, diabetes mellitus [13–15] and death from coronary heart disease [16]. Moreover, the flavonoid rutin is a pharmacologically active phytochemical and natural antioxidant that has been investigated
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incorporated polyethylene glycol-modified cholesterol ester oxidase. Low-density lipoprotein (LDL) concentrations were calculated by the Friedewald formula. Triacylglycerol concentrations were assayed enzymatically with glycerol kinase after lipoprotein lipase hydrolysis followed by oxidation to dihydroxyacetone phosphate and hydrogen peroxide [20]. The enzymatic activities of alanine aminotransferase (ALT – E.C. 2.6.1.2), aspartate aminotransferase (AST – E.C. 2.6.1.1) and lactate dehydrogenase (LDH – E.C. 1.1.1.27) in serum, liver and heart were measured by the enzymatic method of Reitman and Frankel [21], according to the oxidation rate of NADH during the reaction, which is proportional to enzymatic activities. Hepatic and cardiac (left ventricle) tissues were homogenized in 0.6 M perchloric acid and the concentration of free glucose was determined by the glucose oxidase procedure. The glycogen was then hydrolyzed with amyloglucosidase (Sigma, St. Louis, MO, USA) and the total glucose released was measured [22]. The hepatic triacylglycerol was extracted using the procedure developed by Bligh and Dyer [23]. Part of the sample (approximately 200 mg) was homogenized in chloroform-methanol 2:1 (v/v), with the chloroform layer containing all the lipids and the metabolic layer containing all the nonlipids. The hepatic triacylglycerol was measured as described above for serum, by the method of Soloni [20]. Homogenates were prepared on ice at the ratio 200 mg hepatic and cardiac (left ventricle) tissues per 5 mL of 0.01 M phosphate buffer (pH 7.4) in a Potter-Elvehjem type homogenizer. The homogenates were centrifuged at 12,000 g for 20 min at 48 C [24], and the resultant supernatant was used for determination of total protein and the activities of ALT and dehydrogenase lactate. Statistical differences between groups were assessed by analysis of variance (ANOVA) followed by Tukey’s test. P values less than 0.05 were considered statistically significant [25]. All the results were expressed as means S.D. for 10 animals in each group.
for its possible role in protection against and prevention of pathologies [17,18]. Therefore, the purpose of the present study was to examine the influence of the flavonoid rutin on the biochemical parameters and activities of some enzymes in serum and in the hepatic and cardiac tissues of STZ-induced diabetic rats. 2. Materials and methods Forty adult male Wistar rats weighing 300–350 g were used in the experiment. The rats were housed in polypropylene cages and maintained under standard conditions (12 h light/12 h dark cycle; 25 3oC; 60 5% humidity). The investigation conformed to the principles and guidelines of the Canadian Council on Animal Care as outlined in Guide to the Care and Use of Experimental Animals and was approved by the Ethics Committee for Conduct of Animal Studies at the Institute of Biosciences, Sa˜o Paulo State University. Diabetes was induced by intraperitoneal injection of STZ (Sigma, St. Louis, MO, USA) at the dose of 60 mg kg-1 body weight, dissolved in 0.01 M citrate buffer (pH 4.5). Forty-eight hours after STZ administration, blood glucose was measured by glucometer (Boehringer Mannheim, Eli Lilly Ltd., Sa˜o Paulo, Brazil). Only STZtreated rats with glycemia > 250 mg dL-1 were considered diabetic and included in the study. Seven days after STZ injection, the rutin (Sigma, St. Louis, MO, USA) was dissolved in propylenglycol as a vehicle and injected intraperitoneally to rats once a week at the dose of 50 mg kg-1 body weight, for 45 days. A total of 40 rats (n = 10) were randomly divided into four experimental groups (n = 10) as follows: Group I: normal rats treated with water and fed ad libitum; Group II: normal rats treated with rutin and water, and fed ad libitum; Group III: diabetic rats treated with water and fed ad libitum; Group IV: diabetic rats treated with rutin and water, and fed ad libitum. The food (g) and water (mL) intakes of individual rats were measured daily. Body weights (g) were evaluated each week. At the end of experimental period (45 days), rats fasted for 12 h then were sacrificed by cervical decapitation and fasting blood samples were collected. Serum was separated in a centrifuge at 6000 rpm for 15 min. Also, a portion (200 mg) of the liver and heart tissues were dissected out immediately and washed with ice-cold saline solution and kept at –70oC until analyzed. Biochemical parameters were measured in serum samples with a spectrophotometer from Pharmacia Biotech (Ultrospec 2000, Cambridge, England). The analyses were performed with a CELM kit (Modern Laboratory Equipment Company, Sa˜o Paulo, Brazil). Glycemia was determined by the enzymatic method utilizing glucose oxidase and peroxidase. Serum insulin concentration was determined by enzyme immune assay kit (EIA kit, Cayman Chemical, USA), using an ELISA reader (Biotech Instruments, Inc, USA). Total cholesterol levels were measured enzymatically by cholesterol ester/oxidase. High-density lipoprotein (HDL) was measured after precipitation of VLDL and LDL by the sodium phosphotungstate/Mg+2 method, as previously reported by Princen et al. [19], and using an enzymatic colorimetric method that
3. Results Table 1 indicates that diabetes induced a significant increase (p < 0.05) in food and water intake. A significant decrease in body weight gain was observed in STZ-induced diabetic rats (group III) when compared to controls (group I). However, rutin-treated diabetic rats (group IV) showed augmented body weight despite diminished food and water intakes. The insulin serum concentration was significantly lower (p < 0.05) in the diabetic group than in controls. However, serum insulin concentrations were not significantly different between the untreated and treated diabetic rats. The blood glucose increased in STZ-diabetic rats (group III) as compared to normal rats (group I). However, treatment of STZ-diabetic rats with rutin (group IV) significantly reduced the hyperglycemia. The levels of total cholesterol, LDL-cholesterol and triacylglycerol in the serum of the diabetic group treated with rutin (IV) were lower (p < 0.05) than in the untreated group (III). The HDL-cholesterol level was lower in the untreated diabetic rat than the others groups. In the treated diabetic rats (group IV), the levels of triacylglycerol,
Table 1 Body weight gain, food and water intakes of all experimental groups, after 45 days. Groups Parameters
I
II
III
IV
Body weight gain (g) Food intakes (g rat-1 day-1) Water intakes (mL rat-1 day-1)
77.42 20.94c 31.36 3.43a 49.21 7.06a
74.10 22.01c 28.84 5.11a 45.80 6.45a
12.77 9.5a 46.29 4.83b 120.70 14.97c
49.00 11.95b 31.64 3.78a 93.57 12.78b
Values are expressed as means S.D. (n = 10). a,b,cIn each row, means followed by different letter represent significant difference (p < 0.05). Group I: untreated controls; group II: treated controls; group III: untreated diabetics; group IV: treated diabetics.
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Table 2 Serum biochemical parameters of all experimental groups, after 45 days. Biochemical parameters
Groups
Insulin (ng/ml) Glucose (mg 100mL-1) Total cholesterol (mg 100mL-1) LDL-cholesterol (mg 100mL-1) HDL-cholesterol (mg 100mL-1) Triacylglycerols (mg 100mL-1)
I
II
III
IV
2.45 0.67b 98.93 13.69a 89.86 5.98a 36.37 5.62a 35.51 3.40b 99.73 5.76a
2.09 0.52b 104.41 11.90a 93.39 5.14a 36.91 5.06a 37.81 5.65b 99.97 5.64a
0.59 0.18a 397.65 57.45c 172.47 5.29b 112.24 13.77b 25.73 4.92a 184.22 8.51b
0.82 0.21a 149.72 30.31b 105.57 9.04a 53.60 10.97a 36.03 6.11b 107.90 6.58a
Values are expressed as means S.D. (n = 10). a,b,cIn each row, means followed by different letter represent significant difference (p < 0.05). Group I: untreated controls; group II: treated controls; group III: untreated diabetics; group IV: treated diabetics.
Table 3 Enzymatic activities in serum, liver and heart of all experimental groups, after 45 days. Enzymatic activity
Groups
Serum (IU/L)
I
II
III
IV
ALT AST LDH
75.00 6.15a 124.58 6.29a 94.22 4.28a
70.16 8.30a 117.76 4.05a 134.35 6.68b
160.35 21.53c 209.26 20.98c 251.65 11.57d
96.87 4.72b 164.84 10.35b 158.60 9.51c
Liver (IU/mg protein) AST ALT
25.03 1.74b 20.61 1.50b
23.51 1.75b 19.47 1.70b
10.28 2.83a 11.86 1.08a
27.07 1.43b 17.06 1.42b
Heart (IU/mg protein) AST LDH
40.11 2.05c 60.65 9.11c
38.43 2.08c 61.47 12.02c
21.23 2.41a 34.87 5.90a
30.91 3.85b 51.08 6.47b
Values are expressed as means S.D. (n = 10). ALT: alanine aminotransferase; AST: aspartate aminotransferase; LDH: lactate dehydrogenase. a,b,cIn each row, means followed by different letter represent significant difference (p < 0.05). Group I: untreated controls; group II: treated controls; group III: untreated diabetics; group IV: treated diabetics.
Table 4 Liver weight and hepatic triacylglycerols and glycogen after 45 days. Biochemical parameters
Liver weight (% body weight) Triacylglycerols (mg g tissue-1) Glycogen (mg g tissue-1)
Groups I
II
III
IV
4.45 1.28a 9.61 0.60a 20.41 1.16b
4.22 1.90a 10.30 0.97a 18.15 0.87b
9.83 1.83b 22.67 1.46c 14.62 0.70a
5.13 1.66a 14.50 1.45b 19.17 1.74b
Values are expressed as means S.D. (n = 10).a,b,cIn each row, means followed by different letter represent significant difference (p < 0.05). Group I: untreated controls; group II: treated controls; group III: untreated diabetics; group IV: treated diabetics.
cholesterol and LDL cholesterol recovered to control values while HDL cholesterol was elevated (Table 2). In STZ-diabetic rats the activities of serum ALT, AST and LDH were significantly increased (p < 0.05). The administration of rutin to STZ-diabetics rats decreased the activities of ALT, AST and LDH. However, the serum ALT, AST and LDH did not return to the basal level compared to untreated controls (group I) (Table 3). Hepatic ALT and AST activities were significantly diminished in STZdiabetic rats. In contrast, the activities of ALT and AST were significantly (p < 0.05) augmented in the liver tissue of diabetics administered rutin (group IV) in relation to the untreated diabetic rats (group III). Cardiac tissue LDH and AST activities were significantly reduced (p < 0.05) in untreated diabetic rats. However, in diabetic rats, rutin caused a rise in cardiac activities of AST and LDH (Table 3).
The present study showed that the dietary rutin led to significant improvement in the lipid metabolism parameters (triacylglycerols) in the liver tissue of diabetic rats (group IV). In diabetic specimens, rutin reduced the rise in hepatic triacylglycerol. In addition, hepatic glycogen increased significantly in treated diabetic rats (group IV) compared to untreated diabetics (group III). The liver weights (% body weight) among untreated diabetic rats (group III) were higher than those of the non-diabetic controls (group I) (Table 4). Cardiac tissue glycogen concentration was significantly higher in treated diabetic rats (group IV) than in untreated diabetics (group III), but this concentration did not return to the basal level in relation to controls. The cardiac triacylglycerol levels in untreated diabetic rats (group III) were significantly higher than those of control group. Rutin prevented STZ-induced elevation of triacylglycerols in the cardiac tissue (Table 5).
Table 5 Cardiac triacylglycerols and glycogen after 45 days. Biochemical parameters
Triacylglycerols (mg g tissue-1) Glycogen (mg g tissue-1)
Groups I
II
III
IV
0.52 0.04a 10.61 1.38c
0.63 0.05a 11.26 1.12c
0.89 0.012b 5.28 0.64a
0.68 0.04a 7.42 0.97b
Values are expressed as means S.D. (n = 10).a,b,cIn each row, means followed by different letter represent significant difference (p < 0.05). Group I: untreated controls; group II: treated controls; group III: untreated diabetics; group IV: treated diabetics.
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4. Discussion and conclusion As expected from previous studies, STZ is cytotoxic to ß-cells and can be used to induce experimental diabetes in rodents [26,27]. When animals are injected with STZ, it induces symptoms of insulin-dependent diabetes mellitus (type 1), i.e., hyperglycemia, polydipsia, depression of body mass gain, augmented food and water intakes and diminished insulin concentration, as previously observed by Al-Awwadi et al. [28]. The body weight decrease in diabetic rats suggests that the loss or degradation of structural proteins may be due to an unavailability of carbohydrates for utilization as an energy source in diabetes, whereas structural proteins are known to contribute to the body weight [11,29]. Also, the decrease in adipose tissue (lipolysis – hydrolysis of triacilglycerols) may contribute to reduction in body weight. The present study has demonstrated diminutions in serum glucose concentration and food and water intakes, and an increase in the body weight gain in STZ-diabetic rats treated with rutin (50 mg kg-1) (Table 2). This could be the result of improved glycemic control produced by rutin. The effect of lowering serum glucose in the absence of a significant change in serum insulin concentration suggests that rutin treatment may involve an insulin-independent mechanism. Rutin may be producing its hypoglycemic effect by means of extrapancreatic action, possibly by stimulating glucose utilization in extrahepatic tissues [30,31]. A strong relationship has been shown between flavonoids and glucose metabolism. This decrease in the glucose concentration is in part explained by an effect of rutin on the metabolism and in part by the direct action of rutin on cell membranes. In addition, flavonoids exert their effect either by promoting the entry of glucose into cells, thus stimulating glycolytic enzymes and glycogenic enzymes (augmenting glucose storage in the liver – up-regulated glycogenesis) and reducing glycogen breakdown (down-regulated glycogenolysis) [11] and depressing gluconeogenic enzymes or by inhibiting the glucose-6-phosphatase in the liver, consequently reducing the release of glucose in the blood [32]. Alterations in plasma lipoprotein metabolism are common in diabetes and tend to exaggerate any preexisting tendencies towards elevated lipid levels [33]. Diabetes is associated with atherosclerosis [34] and dyslipidemia [35,36], with elevated levels of total and LDL, cholesterol, elevated triacylglycerols and diminished levels of HDL cholesterol [8]. A number of reports have suggested that these compounds may also influence atherogenesis by their effect on lipid and lipoprotein metabolism [37,38]. The rise in serum triacylglycerols, cholesterol and LDL-cholesterol levels in the present study indicate derangement of lipid metabolism and increased incidence of cardiac dysfunction in diabetic rats. Elevation of serum lipids indicates either the defective removal or overproduction (or both) of one or more lipoproteins [39]. The serum levels of triacylglycerols, total cholesterol and LDLcholesterol in the rutin-treated diabetic rat group (IV) were lower (p < 0.05) than those in untreated diabetics (group III). Flavonoids decreased blood levels of triacyglycerols and total cholesterol [40]. Rutin is a potent inhibitor of HMG-CoA reductase and also beneficial for lowering serum cholesterol levels [41]. Consequently, these results indicate that administration of rutin facilitates lipid metabolism in diabetic rats. It has been suggested that dietary flavonoids may be anti-atherogenic agents. In this context, research by Raanan et al. [7] have shown the beneficial effect of metabolic control on serum lipids and oxidative stress in patients with type 1 diabetes, indicating that such control reduces cardiovascular risk in these patients.
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LDL levels may decrease due to the reduction of VLDL and the increase of hepatic depuration of LDL precursors [42]. In nonhuman primates, dietary genistein significantly reduces plasma LDL and VLDL cholesterol levels [37]. Table 2 shows that rutin administration significantly augmented serum HDL cholesterol in rats with STZ-induced diabetes. This finding is advantageous since HDL-cholesterol is responsible for the transportation of cholesterol from peripheral tissues to the liver for metabolization. Rutin thus has the potential to prevent the formation of atherosclerosis and coronary heart disease, two secondary complications of severe diabetes mellitus [43]. It was found that diabetes raises the serum activity levels of the liver enzymes AST, ALT and LDH (Table 3). Elevated activities of serum aminotransferases are a common sign of liver and cardiovascular diseases and are observed more frequently among people with diabetes than in the general population [44]. Such alterations of transaminase activity in the tissues are explicable in terms of energy metabolism, as these enzymes play a role in gluconeogenesis [45]. The elevation in alanine aminotransferase found in liver tissue of STZ-induced diabetic rats corroborates earlier findings that attribute the increased gluconeogenesis and ketogenesis observed in diabetes to the high activity of transaminases [46]. In our study, treatment with rutin significantly decreased the elevated transaminase activity and is involved in the interconversion of metabolic intermediates in relation to energy metabolism and gluconeogenesis [47]. The hyperglycemia in patients with type 1 diabetes is a consequence of excessive or insufficient suppression of gluconeogenesis (synthesis of glucose from lactate and gluconeogenic amino acids, which results from a stimulated cycle activity and enhanced glucose-alanine cycle turnover) or glycogenolysis, either of the two components of endogenous glucose production, or a combination of both [48,49]. The increase in the activities of serum AST, ALT and LDH indicated that diabetes may induce hepatic dysfunction [50]. Therefore, the elevation in serum AST and ALT activities may be due mainly to the leakage of these enzymes from the liver cytosol into the bloodstream [51], which reflects the hepatotoxicity inherent in diabetes. The reduction in liver enzyme activities is due primarily to leakage of these enzymes into the bloodstream as a result of diabetic toxicity, which leads to the liver damage [50]. However, treatment of the STZ-diabetic group with rutin (group IV) successfully restored the activities of these enzymes to their normal levels. Therefore, the activity changes in serum and tissue enzymes indicate control of gluconeogenesis as confirmed by the diminished serum glucose levels. Furthermore, normal AST and ALT activities indicate that rutin may inhibit the liver and heart damage induced by STZ. Rutin treatment prevented both the diminutions in AST, ALT and LDH activities in the liver and heart and the elevations in these enzymatic activities in serum that were caused by STZ administration. This may be attributed to glucose utilization through the pentose phosphate pathway [52], interfering with mitochondrial respiration chain and promoting the peripheral glucose utilization by enhancing anaerobic glycolysis. Antioxidants inhibit the increase in serum levels of AST and ALT in STZ-treated mice [53]. The present study aimed to examine the protective effect of rutin on the enzymatic changes in rat serum, liver and heart injured by exposure to STZ. Rutin lowered the pathway enzymes in diabetic rats and reversed the metabolic changes in enzyme activities that occurred due to diabetes. This beneficial effect may have resulted primarily from the hypoglycemic potential of dietary flavonoids in diabetes [54]. LDH is the enzyme involved in the final step of anaerobic glycolysis. Increased activity of LDH in diabetes mellitus has been
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reported. The LDH system reflects the NAD+/NADH ratio, indicated by the lactate/pyruvate ratio of hepatocyte cytosol [29]. Rutintreated diabetic rats have presented significantly restored LDH activity. Normal LDH activity is indicative of improved channeling of (pyruvate) glucose by mitochondrial oxidation [29]. Liver weight, expressed as a percentage of body weight, was higher in diabetics (group III) than in the non-diabetic rat group (I and II) on account of STZ-induced hypertrophy of the liver (Table 4). Liver hypertrophy in diabetic rats is due mainly to fat deposition (triacyglycerols – Table 4). These are typical symptoms of diabetic rats [55]. The liver weight for the diabetic rat group administered rutin (group IV) was significantly lower (p < 0.05) than that of the diabetic rat group (III). Rutin may play an important role in ameliorating liver hypertrophy in diabetic rats. In diabetes, less glycogen is stored in the liver and, in compensation levels of AST and ALT are raised to produce alternative glucose precursors [45]. Hepatic glycogen metabolism is one of the processes important to the maintenance of glucose homeostasis [56]. The liver glycogen content in the treated diabetic group (IV) may have contributed to maintaining a liver weight similar to that of the controls rats (I and II). Restoration of hepatic glycogen by rutin (group IV) may be due to inhibition of glucose-6-phosphatase in the liver, thereby preventing conversion of glucose-6-phosphate to glucose [57]. Therefore, glucose-6-phosphate can be converted to glycogen. The pathway of glycogen synthesis in the liver is mainly via gluconeogenesis (indirect pathway) or a glucose phosphorylation step (direct pathway) [58]. Flavonoids play important roles in preventing the progression of hyperglycemia, partly by increasing hepatic glycolysis and glycogen concentration and/or by lowering hepatic gluconeogenesis [14]. This study demonstrates that in diabetic-induced rat model, rutin was able to normalize glycemia. The mechanism by which rutin improves the metabolic status in diabetes is probably its hypolipidemic property. In addition, rutin administration decreased the degree of tissue damage in diabetes as evidenced by the activities of ALT, AST and LDH. Therefore, rutin has a protective effect against the hepatotoxicity and cardiotoxicity produced by STZ-induced diabetes. Acknowledgments This research was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo - 05/55918-6 (FAPESP) and Fundac¸a˜o para o Desenvolvimento da Universidade Estadual Paulista - 00662/04 (FUNDUNESP). References [1] Theriault BR, Thistlethwaite JRJ, Levisetti MG, Wardrip GL, Szot G, Bruce DS. Induction, maintenance, and reversal of streptozotocin-induced insulindependent diabetes mellitus in the juvenile cynomegus monkey (Macaca fascilularis). Transplantation 1999;68:331–7. [2] Duchatelet S, Zucman SC, Laforgue DD, Blanc H, Timsit J, Julier C. FCRL3-169CT functional polymorphism in type 1 diabetes and autoimmunity traits. Biomed Pharmacother 2007;62:153–7. [3] Turina M, Christ CM, Polk HCJ. Diabetes and hyperglycemia: Strict glycemic control. Crit Care Med 2006;34:5291–300. [4] Naziroglu M, Butterworth PJ. Protective effects of moderate exercise with dietary vitamin C and E blood antioxidative defense mechanism in rats with streptozotocin-induced diabetes. Can J Appl Physiol 2005;30:172–85. [5] Brownlee M. The pathobiology of diabetic complications: A unifying mechanism. Diabetes 2005;54:1615–25. [6] Keenoy BMY, Vertommen J, Leeuw I. The effect of flavonoid treatment on the glycation and antioxidant status in type 1 diabetic patient. Diab Nutr Metab Clin Exper 2005;10:477–83. [7] Raanan S, Haifa K, Kaplan M, Tova N, Naim S. Glycemic control in adolescents with type 1 diabetes mellitus improves lipid serum levels and oxidative stress. Pediatr Diabetes 2008;9:104–9. [8] Cullen P, Eckardstein AV, Souris S, Schule H, Assmann G. Dyslipidaemia and cardiovascular risk in diabetes. Diab Obes Metab 1999;1:189–98.
[9] Ceriello A. Post prandial hyperglycemia and diabetes complications: Is it time to treat? Diabetes 2005;54:1–7. [10] Bolza´n AD, Bianchi MS. Genotoxicity of streptozotocin. Mutat Res 2002;512: 121–34. [11] Sarkhail P, Rahmaipour S, Fadyevatan S, Mohammadirad A, Dehghan G, Amin G. Antidiabetic effect of Phlomis anisodonta: Effects on hepatic cells lipid peroxidation and antioxidant enzymes in experimental diabetes. Pharmacol Res 2007;56:261–6. [12] Bonnefont RD. The role of antioxidant micronutrients in the prevention of diabetic complications. Treat Endocrinol 2004;3:41–52. [13] Fedosova NF, Alisievich SV, Lyadov KV, Romanova EP, Rudko IA, Kubatiev AA. Mechanisms underlying diquertin-mediated regulation of neutrophil function in patients with non-insulin-dependent diabetes mellitus. Bull Exp Biol Med 2004;137:143–6. [14] Jung UJ, Lee MK, Jeong KS, Choi MS. The hypoglycemic effects of hesperidin and naringin are partly mediated by hepatic glucose-regulating enzymes in C57BL/ Ksj-db/db mice. J Nutr 2004;134:2499–503. [15] Souza AMF JA´, Lo´pez JA. Insulin or insulin-like studies on unicellular organisms: a review. Braz Arch Biol Tech 2004;47:973–81. [16] Hodgson JM, Puddey IB. Dietary flavonoids and cardiovascular disease: does the emperors have any clothes? J Hypertens 2005;23:1461–3. [17] Aherne SA, Brien NM. Lack of effect of the flavonoids, myricetin, quercetin, and rutin, on repair of H2O2-induced DNA single-strand breaks in caco-2, hep G2, and V79 cells. Nutr Cancer 2000;38:106–15. [18] Schwedhelm E, Maas R, Troots R, Boger R. Clinical pharmacokinetics of antioxidants and their impact on systemic oxidative stress. Clin Pharmacokinet 2003;42:437–59. [19] Princen HMG, Poppel G, Vogelezang C, Buytenhek R. Supplementation with vitamin E but not beta-carotene in vivo protects low-density lipoprotein from lipid peroxidation in vivo: effect of cigarette smoking. Arteriocler Thromb 1992;12:554–62. [20] Soloni FG. Simplified manual micromethod for determination of serum triglycerides. Clin Chem 1971;17:531–4. [21] Reitman S, Frankel SA. Colorimetric method for the determination of serum glutamic-oxaloacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957;28:56–63. [22] Roehring KJB, Allred JB. Direct enzymatic produce for the determination of liver glycogen. Anal Biochem 1974;58:414–21. [23] Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959;37:911–7. [24] Pereira B, Costa-Rosa LFBP, Bechara ELK, Newsholme P. Changes in the TBARS content and superoxide dismutase, catalase and glutathione peroxidase activities in the lymphoid organs and skeletal muscles of adrenomedullated rats. Braz J Med Biol Res 1998;31:827–33. [25] Zar JH. Biostatistical Analysis. New Jersey: Prentice; 1996 [718 p]. [26] Coskun O, Kanter M, Korkmaz A, Oter S. Quercetin, a flavonoid antioxidant, prevents and protects streptozotocin-induced oxidative stress and b-cell damage in rat pancreas. Pharmacol Res 2005;51:17–123. [27] Leibovici M, Kanarek RB. Diet selection improves morphine’s antinociceptive actions in rats with streptozotocin-induced diabetes. Behav Neurosci 2007;121:965–75. [28] Al-Awwadi N, Azay J, Poucheret P, Cassanas G, Krosniak M, Auger C. Antidiabetic activity of red wine polyphenolic extract, thanol, or both in streptozotocin-treated rats. J Agric Food Chem 2004;52:1008–16. [29] Sekar DS, Sivagnanam K, Subramaian S. Antidiabetic activity of Momordica charantia sedds on streptozotocin induced diabetic rats. Pharmazie 2005;60:383–7. [30] Mezei O, Banz WJ, Steger RW, Peluso MR, Winters TA, Shay N. Soy isoflavones exert antidiabetic and hypolipidemic effects through the PPAR pathways in obese zucker rats and murine RAW 264.7 cells. Biochem Mol Act Nutr 2003;58:1238–43. [31] Pinent M, Blay M, Blade´ MC, Salvado´ MJ, Arola L. Grape seed-derived procyanidins have an antihyperglycemic effect in streptozotocin-induced diabetic rats and insulinomimetic activity in insulin-sensitive cell lines. Endocrinology 2004;145:4985–90. [32] Naik SR, Fliho JMB, Dhuley JN, Deshmukh A. Probable mechanism of hypoglycaemic activity of bassic acid, a natural product isolated from Bumelia sartorum. J Ethnopharmacol 1999;33:37–44. [33] Merzouk S, Hichami A, Sari A, Madani S, Habane SN, Khan NA. Impaired oxidant/antioxidant status and LDL-fatty acid composition are associated with increased susceptibility to peroxidation of LDL in diabetic patients. Gen Physiol Biophys 2004;23:387–99. [34] Stout RW. Diabetes and atherosclerosis. Biomed Pharmacother 1993;47:1–2. [35] Adiels M, Olofsson SO, Taskinem MR, Boren J. Diabetic dyslipidaemia. Curr Opin Lipidol 2006;17:238–46. [36] Martin A, Sven-Olof O, Marja RT, Jan B. Diabetic dyslipidaemia. Cur Opin Lipidol 2006;17:238–46. [37] Wilcox LJ, Borradaile MN, Huff MW. Antiatherogenic properties of naringerin, acitrus flavonoids. Cardiovas Drug Rev 2000;17:160–78. [38] Tomkin GH. The effect of antidiabetic drugs on genes regulating lipid metabolism. Nutr Metab 2009;20:10–6. [39] Akula A, Kota MK, Gopisetty SG. Biochemical, histological and echocardiographic changes during experimental cardiomyopathy in STZ-induced diabetic rats. Pharmacol Res 2003;48:429–35. [40] Imai K, Nakachi K. Cross-sectional study of effects of drinking green tea on cardiovascular and liver diseases. BMJ 1995;310:693–6.
A.A.H. Fernandes et al. / Biomedicine & Pharmacotherapy 64 (2010) 214–219 [41] Bok SH, Lee SH, Park YB. Plasma and hepatic cholesterol and hepatic activities of 3-hydroxy-3-methyl-glutaryl-CoA reductase and acyl CoA: cholesterol transferase are lower in rats fed citrus peel extractor a mixture of citrus bioflavonoids. J Nutr 1999;129:1182–5. [42] Knekt P, Kumpulainen J, Jarvinen R. Flavonoid intake and risk of chronic diseases. Am J Clin Nutr 2002;76:560–8. [43] Nigdikar SV, Williams NR, Griffin BA, Howard A. Consumption of red wine polyphenols reduces the susceptibility of low-density lipoproteins to oxidation in vivo. Am J Clin Nur 1998;68:258–65. [44] Arkkila PE, Koskinen PJ, Kantola IM, Viikari JS. Diabetic complications are associated with liver enzyme activities in people with type 1 diabetes. Diabetes Res Clin Pract 2001;52:113–8. [45] Mori MD, Baviera AM, Ramalho LTO, Vendramini RC, Brunetti IL, Pepato MT. Temporal response pattern of biochemical analytes in experimental diabetes. Biotechnol Appl Biochem 2003;38:183–91. [46] Punitha ISR, Rajendran K, Shirwaikar A. Alcoholic stem extract of Coscinium fenestratum regulates carbohydrate metabolism and improves antioxidant status in streptozotocin-nicotinamide induced diabetic rats. Evid Based Complement Altern Med 2005;2:375–81. [47] Babu PS, Srinivasan K. Renal lesions in streptozotocin-induced diabetic rats maintained on onion and capsaicin containing diets. J Nutr Biochem 1999;10:477–83. [48] Boden G, Cheung P, Hombo C. Effects of acute insulin excess and deficiency on gluconeogenesis and glycogenolysis in type 1 diabetes. Diabetes 2003;52: 133–7. [49] Barth E, Albuszies G, Baumgart K, Matejovic M, Wachter U, Vogt J. Glucose metabolism and catecholamines. Crit Care Med 2007;35:508–18.
219
[50] Demerdash FM, Yousef MI, El-Naga ANI. Biochemical study on the hypoglycemic effects of onion and garlic in alloxan-induced diabetic rats. Food Chem Toxicol 2005;43:57–63. [51] Kumar G, Murugesan AG, Rajasekara M. Effect of Helicteres isora bark extract on blood glucose and hepatic enzymes in experimental diabetes. Pharmazie 2006;61:353–5. [52] Ugochukwu NH, Babady NE. Antihyperglycaemic effect of aqueous and ethanolic extract of Gongronema latifolium levels of glucose and glycogen metabolism in liver of normal and STZ-induced diabetic rats. Life Sci 2003; 73:1924–38. [53] Imaeda A, Kaneko T, Aoki T, Kondo Y, Nakamura N, Nagase H. Antioxidative effects of fluvastatin and its metabolites against DNA damage in streptozotocin treated mice. Food Chem Toxicol 2002;40:1415–22. [54] Bolkent S, Yanardag R, Karabulut BO, Ozsoy SO. The morphological and biochemical effects of glibornuride on rat in experimental diabetes. Hum Exper Toxicol 2004;23:257–64. [55] Sugiura M, Ohshima M, Ogawa K, Yano M. Chronic administration of Satsuma mandarin fruit improves oxidative stress in streptozotocin-induced diabetic rat liver. Biol Pharm Bul 2006;29:588–91. [56] Hers HG. The control glycogen metabolism in the liver. Rev Biochem 1976; 45:167–89. [57] Wittenstein B, Klein M, Finckh B, Ullrich K, Kohlschutter A. Plasma antioxidants in pediatric patients with glycogen storage disease, diabetes mellitus, and hypercholesterolemia. Free Radic Biol Med 2002; 33:103–10. [58] Mc Garry JD, Kuwajima M, Newgard CB, Foster DW. From dietary glucose to liver glycogen: the full circle round. Annu Rev Nutr 1987;7:51–73.
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Influence of rutin treatment on biochemical alterations in experimental diabetes Ana Ange´lica Henrique Fernandes a,*, Ethel Lourenzi Barbosa Novelli a, Katashi Okoshi b, Marina Politi Okoshi b, Bruno Paulino Di Muzio a, Julliano F. Campos Guimara˜es b, Ary Fernandes Junior c a b c
Department of Chemistry and Biochemistry, Institute of Biosciences, Sa˜o Paulo State University, UNESP, Botucatu, CEP 18618-000, Sa˜o Paulo, Brazil Department of Clinical and Cardiology, Faculty of Medicine, Sa˜o Paulo State University, UNESP, Botucatu, Sa˜o Paulo, Brazil Department of Microbiology and Immunology, Institute of Biosciences, Sa˜o Paulo State University, UNESP, Botucatu, Sa˜o Paulo, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 June 2009 Accepted 24 August 2009 Available online 27 October 2009
Dietary antioxidant compounds such as flavonoids may offer some protection against early-stage diabetes mellitus and its complications. Abnormalities in both glucose metabolism and lipid profile constitute one of the most common complications in diabetes mellitus. The present study aimed to evaluate the effect of rutin, through biochemical parameters, on experimental streptozotocin (STZ)induced diabetes in rats. Male Wistar rats were divided into four groups: untreated controls (GI); normal rats receiving rutin (GII); untreated diabetics (GIII); diabetic rats receiving rutin (GIV). STZ was injected at a single dose of 60 mg kg-1 to induce diabetes mellitus. The diabetes resulted in increased serum glucose, cholesterol, triacylglycerols and lipoproteins (LDL and VLDL-cholesterol) but decresed serum HDL-cholesterol and insulin. Rutin (50 mg kg–1) reduced (p < 0.05) blood glucose and improved the lipid profile in STZ-induced diabetic rats. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activities were significantly augmented in serum of STZ-diabetic rats, while these activities were diminished in hepatic and cardiac tissues compared with the control group. Rutin prevents changes in the activities of ALT, AST and LDH in the serum, liver and heart, indicating the protective effect of rutin against the hepatic and cardiac toxicity caused by STZ. Rutin was associated with markedly decreased hepatic and cardiac levels of tryacylglycerols and elevated glycogen. These results suggest that rutin can improve hyperglycemia and dyslipidemia while inhibiting the progression of liver and heart dysfunction in STZ-induced diabetic rats. ß 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Diabetes Dyslipidemia Rutin
1. Introduction Diabetes mellitus is a heterogeneous metabolic disorder characterized by hyperglycemia resulting from defective insulin secretion, resistance to insulin action or both [1,2] and in stimulating glucose uptake in tissues. Type 1 diabetes is the consequence of an autoimmune-mediated destruction of pancreatic b-cells, leading to insulin deficiency [3]. The disease is characterized by disturbance of carbohydrate and lipid metabolism, and is diagnosed by the presence of hyperglycemia [4]. The clinical manifestation of diabetes is associated with the development of certain diabetic complications [5]. In patients with diabetes, the abnormalities in lipid metabolism generally lead to elevated levels of serum lipids and lipoproteins that, in turn, play an important role in the occurrence of premature and severe atherosclerosis [6,7]. Furthermore, type 1 diabetes leads to dyslipidemia with elevated levels of total and low density
* Corresponding author. Tel.: +55 14 38116255; fax: +55 14 38116255. E-mail address: [email protected] (A.A.H. Fernandes). 0753-3322/$ – see front matter ß 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2009.08.007
lipoprotein (LDL) cholesterol, elevated tryacylglicerols and low levels of high density lipoprotein (HDL) cholesterol [8,9]. Since streptozotocin (STZ) has been recognized as toxic to bcells of the Islets of Langerhans, it has been widely utilized to induce type 1 diabetes mellitus with concomitant insulin deficiency [10]. STZ-induced diabetes in rats is one of the animal models of human diabetes mellitus [11]. After insulin treatment became available, evidence emerged suggesting that human diabetes mellitus has a multifactorial etiology. Insulin was and still is the principle hypoglycemic medication used in diabetes mellitus treatment. In order to discover other hypoglycemic agents, many investigations have been performed on traditional medicines to test prospective hypoglycemic natural substances [12]. Natural products used in folk medicine to treat diabetes represent a viable alternative for the control of this disease. Studies have examined the effects of natural flavonoids on physiological and pathological conditions of glucose metabolism, diabetes mellitus [13–15] and death from coronary heart disease [16]. Moreover, the flavonoid rutin is a pharmacologically active phytochemical and natural antioxidant that has been investigated
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incorporated polyethylene glycol-modified cholesterol ester oxidase. Low-density lipoprotein (LDL) concentrations were calculated by the Friedewald formula. Triacylglycerol concentrations were assayed enzymatically with glycerol kinase after lipoprotein lipase hydrolysis followed by oxidation to dihydroxyacetone phosphate and hydrogen peroxide [20]. The enzymatic activities of alanine aminotransferase (ALT – E.C. 2.6.1.2), aspartate aminotransferase (AST – E.C. 2.6.1.1) and lactate dehydrogenase (LDH – E.C. 1.1.1.27) in serum, liver and heart were measured by the enzymatic method of Reitman and Frankel [21], according to the oxidation rate of NADH during the reaction, which is proportional to enzymatic activities. Hepatic and cardiac (left ventricle) tissues were homogenized in 0.6 M perchloric acid and the concentration of free glucose was determined by the glucose oxidase procedure. The glycogen was then hydrolyzed with amyloglucosidase (Sigma, St. Louis, MO, USA) and the total glucose released was measured [22]. The hepatic triacylglycerol was extracted using the procedure developed by Bligh and Dyer [23]. Part of the sample (approximately 200 mg) was homogenized in chloroform-methanol 2:1 (v/v), with the chloroform layer containing all the lipids and the metabolic layer containing all the nonlipids. The hepatic triacylglycerol was measured as described above for serum, by the method of Soloni [20]. Homogenates were prepared on ice at the ratio 200 mg hepatic and cardiac (left ventricle) tissues per 5 mL of 0.01 M phosphate buffer (pH 7.4) in a Potter-Elvehjem type homogenizer. The homogenates were centrifuged at 12,000 g for 20 min at 48 C [24], and the resultant supernatant was used for determination of total protein and the activities of ALT and dehydrogenase lactate. Statistical differences between groups were assessed by analysis of variance (ANOVA) followed by Tukey’s test. P values less than 0.05 were considered statistically significant [25]. All the results were expressed as means S.D. for 10 animals in each group.
for its possible role in protection against and prevention of pathologies [17,18]. Therefore, the purpose of the present study was to examine the influence of the flavonoid rutin on the biochemical parameters and activities of some enzymes in serum and in the hepatic and cardiac tissues of STZ-induced diabetic rats. 2. Materials and methods Forty adult male Wistar rats weighing 300–350 g were used in the experiment. The rats were housed in polypropylene cages and maintained under standard conditions (12 h light/12 h dark cycle; 25 3oC; 60 5% humidity). The investigation conformed to the principles and guidelines of the Canadian Council on Animal Care as outlined in Guide to the Care and Use of Experimental Animals and was approved by the Ethics Committee for Conduct of Animal Studies at the Institute of Biosciences, Sa˜o Paulo State University. Diabetes was induced by intraperitoneal injection of STZ (Sigma, St. Louis, MO, USA) at the dose of 60 mg kg-1 body weight, dissolved in 0.01 M citrate buffer (pH 4.5). Forty-eight hours after STZ administration, blood glucose was measured by glucometer (Boehringer Mannheim, Eli Lilly Ltd., Sa˜o Paulo, Brazil). Only STZtreated rats with glycemia > 250 mg dL-1 were considered diabetic and included in the study. Seven days after STZ injection, the rutin (Sigma, St. Louis, MO, USA) was dissolved in propylenglycol as a vehicle and injected intraperitoneally to rats once a week at the dose of 50 mg kg-1 body weight, for 45 days. A total of 40 rats (n = 10) were randomly divided into four experimental groups (n = 10) as follows: Group I: normal rats treated with water and fed ad libitum; Group II: normal rats treated with rutin and water, and fed ad libitum; Group III: diabetic rats treated with water and fed ad libitum; Group IV: diabetic rats treated with rutin and water, and fed ad libitum. The food (g) and water (mL) intakes of individual rats were measured daily. Body weights (g) were evaluated each week. At the end of experimental period (45 days), rats fasted for 12 h then were sacrificed by cervical decapitation and fasting blood samples were collected. Serum was separated in a centrifuge at 6000 rpm for 15 min. Also, a portion (200 mg) of the liver and heart tissues were dissected out immediately and washed with ice-cold saline solution and kept at –70oC until analyzed. Biochemical parameters were measured in serum samples with a spectrophotometer from Pharmacia Biotech (Ultrospec 2000, Cambridge, England). The analyses were performed with a CELM kit (Modern Laboratory Equipment Company, Sa˜o Paulo, Brazil). Glycemia was determined by the enzymatic method utilizing glucose oxidase and peroxidase. Serum insulin concentration was determined by enzyme immune assay kit (EIA kit, Cayman Chemical, USA), using an ELISA reader (Biotech Instruments, Inc, USA). Total cholesterol levels were measured enzymatically by cholesterol ester/oxidase. High-density lipoprotein (HDL) was measured after precipitation of VLDL and LDL by the sodium phosphotungstate/Mg+2 method, as previously reported by Princen et al. [19], and using an enzymatic colorimetric method that
3. Results Table 1 indicates that diabetes induced a significant increase (p < 0.05) in food and water intake. A significant decrease in body weight gain was observed in STZ-induced diabetic rats (group III) when compared to controls (group I). However, rutin-treated diabetic rats (group IV) showed augmented body weight despite diminished food and water intakes. The insulin serum concentration was significantly lower (p < 0.05) in the diabetic group than in controls. However, serum insulin concentrations were not significantly different between the untreated and treated diabetic rats. The blood glucose increased in STZ-diabetic rats (group III) as compared to normal rats (group I). However, treatment of STZ-diabetic rats with rutin (group IV) significantly reduced the hyperglycemia. The levels of total cholesterol, LDL-cholesterol and triacylglycerol in the serum of the diabetic group treated with rutin (IV) were lower (p < 0.05) than in the untreated group (III). The HDL-cholesterol level was lower in the untreated diabetic rat than the others groups. In the treated diabetic rats (group IV), the levels of triacylglycerol,
Table 1 Body weight gain, food and water intakes of all experimental groups, after 45 days. Groups Parameters
I
II
III
IV
Body weight gain (g) Food intakes (g rat-1 day-1) Water intakes (mL rat-1 day-1)
77.42 20.94c 31.36 3.43a 49.21 7.06a
74.10 22.01c 28.84 5.11a 45.80 6.45a
12.77 9.5a 46.29 4.83b 120.70 14.97c
49.00 11.95b 31.64 3.78a 93.57 12.78b
Values are expressed as means S.D. (n = 10). a,b,cIn each row, means followed by different letter represent significant difference (p < 0.05). Group I: untreated controls; group II: treated controls; group III: untreated diabetics; group IV: treated diabetics.
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Table 2 Serum biochemical parameters of all experimental groups, after 45 days. Biochemical parameters
Groups
Insulin (ng/ml) Glucose (mg 100mL-1) Total cholesterol (mg 100mL-1) LDL-cholesterol (mg 100mL-1) HDL-cholesterol (mg 100mL-1) Triacylglycerols (mg 100mL-1)
I
II
III
IV
2.45 0.67b 98.93 13.69a 89.86 5.98a 36.37 5.62a 35.51 3.40b 99.73 5.76a
2.09 0.52b 104.41 11.90a 93.39 5.14a 36.91 5.06a 37.81 5.65b 99.97 5.64a
0.59 0.18a 397.65 57.45c 172.47 5.29b 112.24 13.77b 25.73 4.92a 184.22 8.51b
0.82 0.21a 149.72 30.31b 105.57 9.04a 53.60 10.97a 36.03 6.11b 107.90 6.58a
Values are expressed as means S.D. (n = 10). a,b,cIn each row, means followed by different letter represent significant difference (p < 0.05). Group I: untreated controls; group II: treated controls; group III: untreated diabetics; group IV: treated diabetics.
Table 3 Enzymatic activities in serum, liver and heart of all experimental groups, after 45 days. Enzymatic activity
Groups
Serum (IU/L)
I
II
III
IV
ALT AST LDH
75.00 6.15a 124.58 6.29a 94.22 4.28a
70.16 8.30a 117.76 4.05a 134.35 6.68b
160.35 21.53c 209.26 20.98c 251.65 11.57d
96.87 4.72b 164.84 10.35b 158.60 9.51c
Liver (IU/mg protein) AST ALT
25.03 1.74b 20.61 1.50b
23.51 1.75b 19.47 1.70b
10.28 2.83a 11.86 1.08a
27.07 1.43b 17.06 1.42b
Heart (IU/mg protein) AST LDH
40.11 2.05c 60.65 9.11c
38.43 2.08c 61.47 12.02c
21.23 2.41a 34.87 5.90a
30.91 3.85b 51.08 6.47b
Values are expressed as means S.D. (n = 10). ALT: alanine aminotransferase; AST: aspartate aminotransferase; LDH: lactate dehydrogenase. a,b,cIn each row, means followed by different letter represent significant difference (p < 0.05). Group I: untreated controls; group II: treated controls; group III: untreated diabetics; group IV: treated diabetics.
Table 4 Liver weight and hepatic triacylglycerols and glycogen after 45 days. Biochemical parameters
Liver weight (% body weight) Triacylglycerols (mg g tissue-1) Glycogen (mg g tissue-1)
Groups I
II
III
IV
4.45 1.28a 9.61 0.60a 20.41 1.16b
4.22 1.90a 10.30 0.97a 18.15 0.87b
9.83 1.83b 22.67 1.46c 14.62 0.70a
5.13 1.66a 14.50 1.45b 19.17 1.74b
Values are expressed as means S.D. (n = 10).a,b,cIn each row, means followed by different letter represent significant difference (p < 0.05). Group I: untreated controls; group II: treated controls; group III: untreated diabetics; group IV: treated diabetics.
cholesterol and LDL cholesterol recovered to control values while HDL cholesterol was elevated (Table 2). In STZ-diabetic rats the activities of serum ALT, AST and LDH were significantly increased (p < 0.05). The administration of rutin to STZ-diabetics rats decreased the activities of ALT, AST and LDH. However, the serum ALT, AST and LDH did not return to the basal level compared to untreated controls (group I) (Table 3). Hepatic ALT and AST activities were significantly diminished in STZdiabetic rats. In contrast, the activities of ALT and AST were significantly (p < 0.05) augmented in the liver tissue of diabetics administered rutin (group IV) in relation to the untreated diabetic rats (group III). Cardiac tissue LDH and AST activities were significantly reduced (p < 0.05) in untreated diabetic rats. However, in diabetic rats, rutin caused a rise in cardiac activities of AST and LDH (Table 3).
The present study showed that the dietary rutin led to significant improvement in the lipid metabolism parameters (triacylglycerols) in the liver tissue of diabetic rats (group IV). In diabetic specimens, rutin reduced the rise in hepatic triacylglycerol. In addition, hepatic glycogen increased significantly in treated diabetic rats (group IV) compared to untreated diabetics (group III). The liver weights (% body weight) among untreated diabetic rats (group III) were higher than those of the non-diabetic controls (group I) (Table 4). Cardiac tissue glycogen concentration was significantly higher in treated diabetic rats (group IV) than in untreated diabetics (group III), but this concentration did not return to the basal level in relation to controls. The cardiac triacylglycerol levels in untreated diabetic rats (group III) were significantly higher than those of control group. Rutin prevented STZ-induced elevation of triacylglycerols in the cardiac tissue (Table 5).
Table 5 Cardiac triacylglycerols and glycogen after 45 days. Biochemical parameters
Triacylglycerols (mg g tissue-1) Glycogen (mg g tissue-1)
Groups I
II
III
IV
0.52 0.04a 10.61 1.38c
0.63 0.05a 11.26 1.12c
0.89 0.012b 5.28 0.64a
0.68 0.04a 7.42 0.97b
Values are expressed as means S.D. (n = 10).a,b,cIn each row, means followed by different letter represent significant difference (p < 0.05). Group I: untreated controls; group II: treated controls; group III: untreated diabetics; group IV: treated diabetics.
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4. Discussion and conclusion As expected from previous studies, STZ is cytotoxic to ß-cells and can be used to induce experimental diabetes in rodents [26,27]. When animals are injected with STZ, it induces symptoms of insulin-dependent diabetes mellitus (type 1), i.e., hyperglycemia, polydipsia, depression of body mass gain, augmented food and water intakes and diminished insulin concentration, as previously observed by Al-Awwadi et al. [28]. The body weight decrease in diabetic rats suggests that the loss or degradation of structural proteins may be due to an unavailability of carbohydrates for utilization as an energy source in diabetes, whereas structural proteins are known to contribute to the body weight [11,29]. Also, the decrease in adipose tissue (lipolysis – hydrolysis of triacilglycerols) may contribute to reduction in body weight. The present study has demonstrated diminutions in serum glucose concentration and food and water intakes, and an increase in the body weight gain in STZ-diabetic rats treated with rutin (50 mg kg-1) (Table 2). This could be the result of improved glycemic control produced by rutin. The effect of lowering serum glucose in the absence of a significant change in serum insulin concentration suggests that rutin treatment may involve an insulin-independent mechanism. Rutin may be producing its hypoglycemic effect by means of extrapancreatic action, possibly by stimulating glucose utilization in extrahepatic tissues [30,31]. A strong relationship has been shown between flavonoids and glucose metabolism. This decrease in the glucose concentration is in part explained by an effect of rutin on the metabolism and in part by the direct action of rutin on cell membranes. In addition, flavonoids exert their effect either by promoting the entry of glucose into cells, thus stimulating glycolytic enzymes and glycogenic enzymes (augmenting glucose storage in the liver – up-regulated glycogenesis) and reducing glycogen breakdown (down-regulated glycogenolysis) [11] and depressing gluconeogenic enzymes or by inhibiting the glucose-6-phosphatase in the liver, consequently reducing the release of glucose in the blood [32]. Alterations in plasma lipoprotein metabolism are common in diabetes and tend to exaggerate any preexisting tendencies towards elevated lipid levels [33]. Diabetes is associated with atherosclerosis [34] and dyslipidemia [35,36], with elevated levels of total and LDL, cholesterol, elevated triacylglycerols and diminished levels of HDL cholesterol [8]. A number of reports have suggested that these compounds may also influence atherogenesis by their effect on lipid and lipoprotein metabolism [37,38]. The rise in serum triacylglycerols, cholesterol and LDL-cholesterol levels in the present study indicate derangement of lipid metabolism and increased incidence of cardiac dysfunction in diabetic rats. Elevation of serum lipids indicates either the defective removal or overproduction (or both) of one or more lipoproteins [39]. The serum levels of triacylglycerols, total cholesterol and LDLcholesterol in the rutin-treated diabetic rat group (IV) were lower (p < 0.05) than those in untreated diabetics (group III). Flavonoids decreased blood levels of triacyglycerols and total cholesterol [40]. Rutin is a potent inhibitor of HMG-CoA reductase and also beneficial for lowering serum cholesterol levels [41]. Consequently, these results indicate that administration of rutin facilitates lipid metabolism in diabetic rats. It has been suggested that dietary flavonoids may be anti-atherogenic agents. In this context, research by Raanan et al. [7] have shown the beneficial effect of metabolic control on serum lipids and oxidative stress in patients with type 1 diabetes, indicating that such control reduces cardiovascular risk in these patients.
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LDL levels may decrease due to the reduction of VLDL and the increase of hepatic depuration of LDL precursors [42]. In nonhuman primates, dietary genistein significantly reduces plasma LDL and VLDL cholesterol levels [37]. Table 2 shows that rutin administration significantly augmented serum HDL cholesterol in rats with STZ-induced diabetes. This finding is advantageous since HDL-cholesterol is responsible for the transportation of cholesterol from peripheral tissues to the liver for metabolization. Rutin thus has the potential to prevent the formation of atherosclerosis and coronary heart disease, two secondary complications of severe diabetes mellitus [43]. It was found that diabetes raises the serum activity levels of the liver enzymes AST, ALT and LDH (Table 3). Elevated activities of serum aminotransferases are a common sign of liver and cardiovascular diseases and are observed more frequently among people with diabetes than in the general population [44]. Such alterations of transaminase activity in the tissues are explicable in terms of energy metabolism, as these enzymes play a role in gluconeogenesis [45]. The elevation in alanine aminotransferase found in liver tissue of STZ-induced diabetic rats corroborates earlier findings that attribute the increased gluconeogenesis and ketogenesis observed in diabetes to the high activity of transaminases [46]. In our study, treatment with rutin significantly decreased the elevated transaminase activity and is involved in the interconversion of metabolic intermediates in relation to energy metabolism and gluconeogenesis [47]. The hyperglycemia in patients with type 1 diabetes is a consequence of excessive or insufficient suppression of gluconeogenesis (synthesis of glucose from lactate and gluconeogenic amino acids, which results from a stimulated cycle activity and enhanced glucose-alanine cycle turnover) or glycogenolysis, either of the two components of endogenous glucose production, or a combination of both [48,49]. The increase in the activities of serum AST, ALT and LDH indicated that diabetes may induce hepatic dysfunction [50]. Therefore, the elevation in serum AST and ALT activities may be due mainly to the leakage of these enzymes from the liver cytosol into the bloodstream [51], which reflects the hepatotoxicity inherent in diabetes. The reduction in liver enzyme activities is due primarily to leakage of these enzymes into the bloodstream as a result of diabetic toxicity, which leads to the liver damage [50]. However, treatment of the STZ-diabetic group with rutin (group IV) successfully restored the activities of these enzymes to their normal levels. Therefore, the activity changes in serum and tissue enzymes indicate control of gluconeogenesis as confirmed by the diminished serum glucose levels. Furthermore, normal AST and ALT activities indicate that rutin may inhibit the liver and heart damage induced by STZ. Rutin treatment prevented both the diminutions in AST, ALT and LDH activities in the liver and heart and the elevations in these enzymatic activities in serum that were caused by STZ administration. This may be attributed to glucose utilization through the pentose phosphate pathway [52], interfering with mitochondrial respiration chain and promoting the peripheral glucose utilization by enhancing anaerobic glycolysis. Antioxidants inhibit the increase in serum levels of AST and ALT in STZ-treated mice [53]. The present study aimed to examine the protective effect of rutin on the enzymatic changes in rat serum, liver and heart injured by exposure to STZ. Rutin lowered the pathway enzymes in diabetic rats and reversed the metabolic changes in enzyme activities that occurred due to diabetes. This beneficial effect may have resulted primarily from the hypoglycemic potential of dietary flavonoids in diabetes [54]. LDH is the enzyme involved in the final step of anaerobic glycolysis. Increased activity of LDH in diabetes mellitus has been
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reported. The LDH system reflects the NAD+/NADH ratio, indicated by the lactate/pyruvate ratio of hepatocyte cytosol [29]. Rutintreated diabetic rats have presented significantly restored LDH activity. Normal LDH activity is indicative of improved channeling of (pyruvate) glucose by mitochondrial oxidation [29]. Liver weight, expressed as a percentage of body weight, was higher in diabetics (group III) than in the non-diabetic rat group (I and II) on account of STZ-induced hypertrophy of the liver (Table 4). Liver hypertrophy in diabetic rats is due mainly to fat deposition (triacyglycerols – Table 4). These are typical symptoms of diabetic rats [55]. The liver weight for the diabetic rat group administered rutin (group IV) was significantly lower (p < 0.05) than that of the diabetic rat group (III). Rutin may play an important role in ameliorating liver hypertrophy in diabetic rats. In diabetes, less glycogen is stored in the liver and, in compensation levels of AST and ALT are raised to produce alternative glucose precursors [45]. Hepatic glycogen metabolism is one of the processes important to the maintenance of glucose homeostasis [56]. The liver glycogen content in the treated diabetic group (IV) may have contributed to maintaining a liver weight similar to that of the controls rats (I and II). Restoration of hepatic glycogen by rutin (group IV) may be due to inhibition of glucose-6-phosphatase in the liver, thereby preventing conversion of glucose-6-phosphate to glucose [57]. Therefore, glucose-6-phosphate can be converted to glycogen. The pathway of glycogen synthesis in the liver is mainly via gluconeogenesis (indirect pathway) or a glucose phosphorylation step (direct pathway) [58]. Flavonoids play important roles in preventing the progression of hyperglycemia, partly by increasing hepatic glycolysis and glycogen concentration and/or by lowering hepatic gluconeogenesis [14]. This study demonstrates that in diabetic-induced rat model, rutin was able to normalize glycemia. The mechanism by which rutin improves the metabolic status in diabetes is probably its hypolipidemic property. In addition, rutin administration decreased the degree of tissue damage in diabetes as evidenced by the activities of ALT, AST and LDH. Therefore, rutin has a protective effect against the hepatotoxicity and cardiotoxicity produced by STZ-induced diabetes. Acknowledgments This research was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo - 05/55918-6 (FAPESP) and Fundac¸a˜o para o Desenvolvimento da Universidade Estadual Paulista - 00662/04 (FUNDUNESP). References [1] Theriault BR, Thistlethwaite JRJ, Levisetti MG, Wardrip GL, Szot G, Bruce DS. Induction, maintenance, and reversal of streptozotocin-induced insulindependent diabetes mellitus in the juvenile cynomegus monkey (Macaca fascilularis). Transplantation 1999;68:331–7. [2] Duchatelet S, Zucman SC, Laforgue DD, Blanc H, Timsit J, Julier C. FCRL3-169CT functional polymorphism in type 1 diabetes and autoimmunity traits. Biomed Pharmacother 2007;62:153–7. [3] Turina M, Christ CM, Polk HCJ. Diabetes and hyperglycemia: Strict glycemic control. Crit Care Med 2006;34:5291–300. [4] Naziroglu M, Butterworth PJ. Protective effects of moderate exercise with dietary vitamin C and E blood antioxidative defense mechanism in rats with streptozotocin-induced diabetes. Can J Appl Physiol 2005;30:172–85. [5] Brownlee M. The pathobiology of diabetic complications: A unifying mechanism. Diabetes 2005;54:1615–25. [6] Keenoy BMY, Vertommen J, Leeuw I. The effect of flavonoid treatment on the glycation and antioxidant status in type 1 diabetic patient. Diab Nutr Metab Clin Exper 2005;10:477–83. [7] Raanan S, Haifa K, Kaplan M, Tova N, Naim S. Glycemic control in adolescents with type 1 diabetes mellitus improves lipid serum levels and oxidative stress. Pediatr Diabetes 2008;9:104–9. [8] Cullen P, Eckardstein AV, Souris S, Schule H, Assmann G. Dyslipidaemia and cardiovascular risk in diabetes. Diab Obes Metab 1999;1:189–98.
[9] Ceriello A. Post prandial hyperglycemia and diabetes complications: Is it time to treat? Diabetes 2005;54:1–7. [10] Bolza´n AD, Bianchi MS. Genotoxicity of streptozotocin. Mutat Res 2002;512: 121–34. [11] Sarkhail P, Rahmaipour S, Fadyevatan S, Mohammadirad A, Dehghan G, Amin G. Antidiabetic effect of Phlomis anisodonta: Effects on hepatic cells lipid peroxidation and antioxidant enzymes in experimental diabetes. Pharmacol Res 2007;56:261–6. [12] Bonnefont RD. The role of antioxidant micronutrients in the prevention of diabetic complications. Treat Endocrinol 2004;3:41–52. [13] Fedosova NF, Alisievich SV, Lyadov KV, Romanova EP, Rudko IA, Kubatiev AA. Mechanisms underlying diquertin-mediated regulation of neutrophil function in patients with non-insulin-dependent diabetes mellitus. Bull Exp Biol Med 2004;137:143–6. [14] Jung UJ, Lee MK, Jeong KS, Choi MS. The hypoglycemic effects of hesperidin and naringin are partly mediated by hepatic glucose-regulating enzymes in C57BL/ Ksj-db/db mice. J Nutr 2004;134:2499–503. [15] Souza AMF JA´, Lo´pez JA. Insulin or insulin-like studies on unicellular organisms: a review. Braz Arch Biol Tech 2004;47:973–81. [16] Hodgson JM, Puddey IB. Dietary flavonoids and cardiovascular disease: does the emperors have any clothes? J Hypertens 2005;23:1461–3. [17] Aherne SA, Brien NM. Lack of effect of the flavonoids, myricetin, quercetin, and rutin, on repair of H2O2-induced DNA single-strand breaks in caco-2, hep G2, and V79 cells. Nutr Cancer 2000;38:106–15. [18] Schwedhelm E, Maas R, Troots R, Boger R. Clinical pharmacokinetics of antioxidants and their impact on systemic oxidative stress. Clin Pharmacokinet 2003;42:437–59. [19] Princen HMG, Poppel G, Vogelezang C, Buytenhek R. Supplementation with vitamin E but not beta-carotene in vivo protects low-density lipoprotein from lipid peroxidation in vivo: effect of cigarette smoking. Arteriocler Thromb 1992;12:554–62. [20] Soloni FG. Simplified manual micromethod for determination of serum triglycerides. Clin Chem 1971;17:531–4. [21] Reitman S, Frankel SA. Colorimetric method for the determination of serum glutamic-oxaloacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957;28:56–63. [22] Roehring KJB, Allred JB. Direct enzymatic produce for the determination of liver glycogen. Anal Biochem 1974;58:414–21. [23] Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959;37:911–7. [24] Pereira B, Costa-Rosa LFBP, Bechara ELK, Newsholme P. Changes in the TBARS content and superoxide dismutase, catalase and glutathione peroxidase activities in the lymphoid organs and skeletal muscles of adrenomedullated rats. Braz J Med Biol Res 1998;31:827–33. [25] Zar JH. Biostatistical Analysis. New Jersey: Prentice; 1996 [718 p]. [26] Coskun O, Kanter M, Korkmaz A, Oter S. Quercetin, a flavonoid antioxidant, prevents and protects streptozotocin-induced oxidative stress and b-cell damage in rat pancreas. Pharmacol Res 2005;51:17–123. [27] Leibovici M, Kanarek RB. Diet selection improves morphine’s antinociceptive actions in rats with streptozotocin-induced diabetes. Behav Neurosci 2007;121:965–75. [28] Al-Awwadi N, Azay J, Poucheret P, Cassanas G, Krosniak M, Auger C. Antidiabetic activity of red wine polyphenolic extract, thanol, or both in streptozotocin-treated rats. J Agric Food Chem 2004;52:1008–16. [29] Sekar DS, Sivagnanam K, Subramaian S. Antidiabetic activity of Momordica charantia sedds on streptozotocin induced diabetic rats. Pharmazie 2005;60:383–7. [30] Mezei O, Banz WJ, Steger RW, Peluso MR, Winters TA, Shay N. Soy isoflavones exert antidiabetic and hypolipidemic effects through the PPAR pathways in obese zucker rats and murine RAW 264.7 cells. Biochem Mol Act Nutr 2003;58:1238–43. [31] Pinent M, Blay M, Blade´ MC, Salvado´ MJ, Arola L. Grape seed-derived procyanidins have an antihyperglycemic effect in streptozotocin-induced diabetic rats and insulinomimetic activity in insulin-sensitive cell lines. Endocrinology 2004;145:4985–90. [32] Naik SR, Fliho JMB, Dhuley JN, Deshmukh A. Probable mechanism of hypoglycaemic activity of bassic acid, a natural product isolated from Bumelia sartorum. J Ethnopharmacol 1999;33:37–44. [33] Merzouk S, Hichami A, Sari A, Madani S, Habane SN, Khan NA. Impaired oxidant/antioxidant status and LDL-fatty acid composition are associated with increased susceptibility to peroxidation of LDL in diabetic patients. Gen Physiol Biophys 2004;23:387–99. [34] Stout RW. Diabetes and atherosclerosis. Biomed Pharmacother 1993;47:1–2. [35] Adiels M, Olofsson SO, Taskinem MR, Boren J. Diabetic dyslipidaemia. Curr Opin Lipidol 2006;17:238–46. [36] Martin A, Sven-Olof O, Marja RT, Jan B. Diabetic dyslipidaemia. Cur Opin Lipidol 2006;17:238–46. [37] Wilcox LJ, Borradaile MN, Huff MW. Antiatherogenic properties of naringerin, acitrus flavonoids. Cardiovas Drug Rev 2000;17:160–78. [38] Tomkin GH. The effect of antidiabetic drugs on genes regulating lipid metabolism. Nutr Metab 2009;20:10–6. [39] Akula A, Kota MK, Gopisetty SG. Biochemical, histological and echocardiographic changes during experimental cardiomyopathy in STZ-induced diabetic rats. Pharmacol Res 2003;48:429–35. [40] Imai K, Nakachi K. Cross-sectional study of effects of drinking green tea on cardiovascular and liver diseases. BMJ 1995;310:693–6.
A.A.H. Fernandes et al. / Biomedicine & Pharmacotherapy 64 (2010) 214–219 [41] Bok SH, Lee SH, Park YB. Plasma and hepatic cholesterol and hepatic activities of 3-hydroxy-3-methyl-glutaryl-CoA reductase and acyl CoA: cholesterol transferase are lower in rats fed citrus peel extractor a mixture of citrus bioflavonoids. J Nutr 1999;129:1182–5. [42] Knekt P, Kumpulainen J, Jarvinen R. Flavonoid intake and risk of chronic diseases. Am J Clin Nutr 2002;76:560–8. [43] Nigdikar SV, Williams NR, Griffin BA, Howard A. Consumption of red wine polyphenols reduces the susceptibility of low-density lipoproteins to oxidation in vivo. Am J Clin Nur 1998;68:258–65. [44] Arkkila PE, Koskinen PJ, Kantola IM, Viikari JS. Diabetic complications are associated with liver enzyme activities in people with type 1 diabetes. Diabetes Res Clin Pract 2001;52:113–8. [45] Mori MD, Baviera AM, Ramalho LTO, Vendramini RC, Brunetti IL, Pepato MT. Temporal response pattern of biochemical analytes in experimental diabetes. Biotechnol Appl Biochem 2003;38:183–91. [46] Punitha ISR, Rajendran K, Shirwaikar A. Alcoholic stem extract of Coscinium fenestratum regulates carbohydrate metabolism and improves antioxidant status in streptozotocin-nicotinamide induced diabetic rats. Evid Based Complement Altern Med 2005;2:375–81. [47] Babu PS, Srinivasan K. Renal lesions in streptozotocin-induced diabetic rats maintained on onion and capsaicin containing diets. J Nutr Biochem 1999;10:477–83. [48] Boden G, Cheung P, Hombo C. Effects of acute insulin excess and deficiency on gluconeogenesis and glycogenolysis in type 1 diabetes. Diabetes 2003;52: 133–7. [49] Barth E, Albuszies G, Baumgart K, Matejovic M, Wachter U, Vogt J. Glucose metabolism and catecholamines. Crit Care Med 2007;35:508–18.
219
[50] Demerdash FM, Yousef MI, El-Naga ANI. Biochemical study on the hypoglycemic effects of onion and garlic in alloxan-induced diabetic rats. Food Chem Toxicol 2005;43:57–63. [51] Kumar G, Murugesan AG, Rajasekara M. Effect of Helicteres isora bark extract on blood glucose and hepatic enzymes in experimental diabetes. Pharmazie 2006;61:353–5. [52] Ugochukwu NH, Babady NE. Antihyperglycaemic effect of aqueous and ethanolic extract of Gongronema latifolium levels of glucose and glycogen metabolism in liver of normal and STZ-induced diabetic rats. Life Sci 2003; 73:1924–38. [53] Imaeda A, Kaneko T, Aoki T, Kondo Y, Nakamura N, Nagase H. Antioxidative effects of fluvastatin and its metabolites against DNA damage in streptozotocin treated mice. Food Chem Toxicol 2002;40:1415–22. [54] Bolkent S, Yanardag R, Karabulut BO, Ozsoy SO. The morphological and biochemical effects of glibornuride on rat in experimental diabetes. Hum Exper Toxicol 2004;23:257–64. [55] Sugiura M, Ohshima M, Ogawa K, Yano M. Chronic administration of Satsuma mandarin fruit improves oxidative stress in streptozotocin-induced diabetic rat liver. Biol Pharm Bul 2006;29:588–91. [56] Hers HG. The control glycogen metabolism in the liver. Rev Biochem 1976; 45:167–89. [57] Wittenstein B, Klein M, Finckh B, Ullrich K, Kohlschutter A. Plasma antioxidants in pediatric patients with glycogen storage disease, diabetes mellitus, and hypercholesterolemia. Free Radic Biol Med 2002; 33:103–10. [58] Mc Garry JD, Kuwajima M, Newgard CB, Foster DW. From dietary glucose to liver glycogen: the full circle round. Annu Rev Nutr 1987;7:51–73.