- Email: [email protected]
Modulatory effect of S-allylcysteine on glucose metabolism in streptozotocin induced diabetic rats
JOURNAL OF FUNCTIONAL FOODS
1 ( 2 0 0 9 ) 3 3 6 –3 4 0
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jff
Modulatory effect of S-allylcysteine on glucose metabolism in streptozotocin induced diabetic rats Ganapathy Saravanana,b,*, Ponnusamy Ponmurugana,c, Gandhipuram Periasamy Senthilkumarb, Thatchinamoorthi Rajarajand a
Research and Development Centre, Bharathiyar University, Coimbatore 641046, Tamilnadu, India Department of Biochemistry, Centre for Biological science, K.S.R. College of Arts and Science, Thokkavadi, Tiruchengode 637215, Tamil Nadu, India c Department of Biotechnology, K.S.R. College of Technology, Thokkavadi, Tiruchengode 637215, Tamil Nadu, India d Department of Biochemistry, School of Biotechnology, Sastra University, Thanjavore, Tamil Nadu, India b
A R T I C L E I N F O
A B S T R A C T
Article history:
The antihyperglycemic effect of S-allylcysteine (SAC) on normal and streptozotocin (STZ)
Received 14 May 2009
diabetic rats was investigated. Diabetes was induced into male albino Wistar rats by intra-
Received in revised form
peritoneal administration of STZ. The SAC was administered orally (150 mg/kg bw) to nor-
4 August 2009
mal and STZ-diabetic rats for 45 days. The diabetic rats showed an increase in levels of
Accepted 1 September 2009
blood glucose. In addition, diabetic rats showed a significant reduction in the activity of
Available online 23 September 2009
hexokinase, glycogen synthase, liver glycogen and an elevation in the activities of enzymes such as glucose-6-phosphatase, fructose-1,6-bisphosphatase and glycogen phosphorylase.
Keywords:
Treatment with SAC significantly decreased blood glucose and. SAC administration to dia-
S-Allylcysteine
betic rats reversed these enzyme activities in a significant manner. Thus, the results show
Streptozotocin
that SAC possesses an antihyperglycemic activity and provide evidence for its traditional
Diabetes
usage in the control of diabetes. Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Diabetes mellitus (DM) is a group of disorders with different etiologies. It is characterized by derangements in carbohydrate, protein and fat metabolism, caused by complete or relative insufficiency of insulin secretion and/or insulin action (Balkau et al., 2000). According to Food and Agriculture Organization (FAO) projections, the prevalence of diabetes is likely to increase by 35% by the year 2025 (Boyle et al., 2001) and approximately, 140 million people worldwide suffer from diabetes. The disease becomes a real problem of public health in the developing countries, where its prevalence is increasing
steadily. Developing countries like India have today become the diabetic capital of the world with over 20 million diabetics and this number is set to increase to 57 million by 2025 which makes it a country with the highest number of diabetics in the world (King et al., 1998). The control of blood glucose in diabetic patients is achieved mainly by the use of oral hypoglycemic/antihyperglycemic agents and insulin. However, all these treatments have limited efficacy and have been reported to be associated with undesirable side effects (Harrower, 1994). In order to overcome the side effects associated with diabetes, interest has been shifted to the use of alternative medicine.
Abbreviations: SAC, S-allylcysteine; STZ, Streptozotocin * Corresponding author: Present address: PG and Research Department of Biochemistry, Centre for Biological science, K.S.R. College of Arts and Science, Thokkavadi, Tiruchengode 637215, Tamil Nadu, India. E-mail address: [email protected] (G. Saravanan). 1756-4646/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jff.2009.09.001
JOURNAL OF FUNCTIONAL FOODS
Medicinal plants and their bioactive constituents have been extensively used as alternative medicine for better control and management of diabetes mellitus. According to world ethnobotanical information reports, almost 800 plants may possess antidiabetic potential (Alarcon-Aguilara et al., 1998). In the past decade, research has been focused on scientific evaluation of traditional drugs of plant origin and screening of more effective and safe hypoglycemic agents has continued to be an important area. In developing countries 80% of populations are using traditional medicine in primary medical care (Grover and Yadav, 2004). However, a large number of herbs are now being used in the management of DM. India has a wealth of medicinally important plants and ancient herbal treatment methods where traditional alternative medicines are popularly practiced among a large segment of its population. Although several medicinal plants have gained importance for the treatment of diabetes mellitus, many remain to be scientifically investigated. Consumption of garlic (Allium sativum, Liliaceae) and its components has been reported to have many medicinal properties including antidiabetic and anti-lipidemic activities (McKenna et al., 2002). S-allylcysteine is a sulphur-containing amino acid derived from garlic, has been reported to have antioxidant (Herrera-Mundo et al., 2006), anticancer (Chu et al., 2007), and neurotrophic (Moriguchi et al., 1997) activities. The present study was undertaken to determine the effect of S-allylcysteine on diabetes-induced changes in hepatic enzyme activities associated with glucose metabolism with a view to elucidating the probable mechanism by which S-allylcysteine reduces blood glucose.
2.4.
1 ( 20 0 9) 3 3 6–34 0
337
Experimental design
After successful induction of experimental diabetes, the rats were divided into four groups each comprising a minimum of six rats. These were: Group 1, control rats; Group 2, Diabetic control rats; Group 3, Diabetic rats administered with SAC (150 mg/kg bw/rat) in aqueous solution orally for 45 days (Saravanan et al., 2009); and Group 4, Diabetic rats administered with glyclazide (5 mg/kg/bw/rat) in aqueous solution orally for 45 days (Pulido et al., 1997). Body weight and blood glucose level measurements were conducted periodically. At the end of the experimental period, rats were fasted overnight, anaesthetized and sacrificed by cervical decapitation. The blood was collected with or without EDTA (ethylenediaminetetraacetic acid) for plasma or serum separation, respectively.
2.5.
Biochemical assays
Glucose levels were estimated by a commercially available glucose kit based using the glucose oxidase method (Sigma Diagnostics, St. Louis, MO). Liver was immediately dissected, washed in ice-cold saline to remove the blood and homogenised in 0.1 M Tris–HCl buffer, pH 7.4. The supernatant was used for enzyme activity assays. Hexokinase, glucose-6-phosphatase and fructose-1,6-bisphosphatase were assayed by the method of Lapeir and Rodnick (2001), Koida and Oda (1959) and Gancedo and Gancedo (1971), respectively. Glycogen synthase and phosphorylase activities were assayed by the method of Leloir and Goldenber (1962) and Cornblath et al. (1963), respectively. Liver glycogen was assayed by the method of Ong and Khoo (2000).
2.
Materials and methods
2.6.
2.1.
Animals
All the grouped data were statistically evaluated with SPSS/ 10.0 software. Hypothesis testing methods included one way analysis of variance (ANOVA) followed by least significant difference (LSD) test; p value of less than 0.05 were considered to indicate statistical significance. All the results were expressed as the mean ± S.D. for six animals in each group.
Male Wister rats of body weight 150–180 g were obtained from Nandha College of Pharmacy, Erode, India. They were kept in the Animal House, Sastra University, Thanjavore, India, on a standard pellet diet (AMRUT, Pune, India) and water ad libitum. The protocol of this study was approved by the Institutional Ethical Committee of Sastra University, Thanjavore, India.
2.2.
Chemicals
SAC (99%) was purchased from LGC Prochem, Bangalore, India. Streptozotocin was purchased from Himedia, Bangalore, India. All other chemicals used were of analytical grade.
2.3.
Induction of diabetes
Diabetes was induced (Kaleem et al., 2006) in overnight fasted adult Wistar strain albino male rats weighing 150–180 g by a single intraperitoneal injection of 55 mg/kg streptozotocin. Streptozotocin (55 mg/kg) was dissolved in 0.1 M citrate buffer (pH 4.5). Hyperglycemia was confirmed by the elevated glucose levels (Above 250 mg/dl) in plasma, determined at 72 h and then on day 7 after injection.
3.
Statistical analysis
Results
Table 1 shows the glucose level of control and experimental group of rats. The blood glucose level in the control rats was significantly increased in diabetic rats. Treatment with SAC as well as glyclazide to diabetic rats elicited significant (p < 0.05) decreases in blood glucose level when compared with diabetic control rats. Table 2 summarizes the levels of hexokinase, glucose-6phosphatase and fructose-6-phosphatase in the control and experimental groups of rats. A significant decrease in hepatic hexokinase level and concomitant increase in glucose-6phosphatase and fructose-6-phosphatase level was observed in STZ-diabetic rats and it was normalized after treatment of SAC and glyclazide. Table 3 shows the hepatic glycogen content, and in the activity of glycogen synthase and glycogen phosphorylase in control and experimental groups of rats. There was a signifi-
338
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Table 1 – Effects of SAC on blood glucose and urine sugar in control and experimental animals. Groups
Blood glucose (mg/dl)
Urine sugar
89.15 ± 1.23 276.41 ± 12.63a,* 96.34 ± 1.47b,* 93.68 ± 1.68b,*
Nil +++ Nil Nil
Control Diabetic control Diabetic + SAC (150 mg/kg) Diabetic + glyclazide Values are mean ± S.D., n = 6. Significantly different from control. b Significantly different from diabetic control. * p < 0.05. a
Table 2 – Effects of SAC on hexokinase, glucose-6-phosphatase and fructose-1,6-bisphosphatase in control and experimental animals. Groups Control Diabetic Diabetic + SAC (150 mg/kg) Diabetic + glyclazide
Hexokinase
Glucose-6-phosphatase
265.41 ± 11.93 136.73 ± 9.42 a,* 252.68 ± 10.12 b,* 256.54 ± 9.63b,*
965.13 ± 46.48 1600.14 ± 120.95 a,* 1042.10 ± 46.88 b,* 1012.22 ± 48.54b,*
Fructose-1,6-bisphosphatase 465.13 ± 22.45 750.65 ± 63.53 a,* 660.31 ± 24.90 b,* 575.16 ± 26.34b,*
Values are mean ± S.D., n = 6. Hexokinase: lmol of glucose phosphorylated/min/mg of protein, glucose-6-phosphatase: lmol of Pi liberated/min/mg of protein, fructose-1,6bisphosphatase: lmol of phosphate liberated/min/mg of protein. a Significantly different from control. b Significantly different from diabetic control. * p < 0.05.
Table 3 – Effects of SAC on glycogen, glycogen synthase and glycogen phosphorylase in control and experimental animals. Groups Control Diabetic Diabetic + SAC (150 mg/kg) Diabetic + glyclazide
Glycogen (mg/g wet tissue) 56.15 ± 2.98 22.40 ± 1.15a,* 50.65 ± 1.95b,* 52.32 ± 1.68b,*
Glycogen synthase 848.54 ± 72.34 462.36 ± 17.98a,* 810.63 ± 66.13b,* 823.95 ± 68.21b,*
Glycogen phosphorylase 610.21 ± 23.15 845.32 ± 33.49a,* 718.10 ± 26.10b,* 712.48 ± 22.14b,*
Values are mean ± S.D., n = 6. Glycogen synthase: lmol of uridine diphosphate formed/h/mg protein glycogen phosphorylase: lmol of phosphate liberated/h/mg protein. a Significantly different from control. b Significantly different from diabetic control. * p < 0.05.
cant reduction in hepatic glycogen content and activity of glycogen synthase and concomitant increase in the activity of glycogen phosphorylase in STZ-diabetic rats when compared with control rats. Oral treatment of SAC and glyclazide tended to bring glycogen content, glycogen synthase and glycogen phosphorylase towards near-normal levels.
4.
Discussion
Streptozotocin is well known for its selective pancreatic islet b-cell cytotoxicity and has been extensively used to induce diabetes mellitus in animals. It interferes with cellular metabolic oxidative mechanisms (Papaccio et al., 2000). Intraperitoneal administration of streptozotocin effectively induced diabetes in normal rats, as observed by hyperglycemia when compared with normal rats. Persistent hyperglycemia, the common characteristic of diabetes can cause most diabetic complications and it is normalized by the action of insulin
(Gayathri and Kannabiran, 2008). In the present study, it was observed that oral administration of SAC could reverse the above mentioned diabetic effect, possibly due to an insulinlike effect of SAC on peripheral tissues, either by promoting glucose uptake and metabolism, or by inhibiting hepatic gluconeogenesis. A number of compounds have also been shown to exert hypoglycemic activity through stimulation of insulin release (Palsamy and Subramanian, 2008). The hypoglycemic potency of SAC was comparable glyclazide, a standard hypoglycemic drug. Glyclazide has long been used to treat diabetes and is known to act by stimulating insulin secretion through action on the pancreatic b-cells (Jeong-Kwon Park et al., 2008). Hexokinase is an insulin-dependent and insulin-sensitive enzyme and are almost completely inhibited or inactivated in diabetic rat liver in the absence of insulin (Gupta et al., 1997). Decreased enzymatic activity of hexokinase has also been reported in diabetic animals, resulting in depletion of liver and muscle glycogen (Murray et al., 2000) . Administration
JOURNAL OF FUNCTIONAL FOODS
of SAC and glyclazide to STZ treated rats resulted in an increased activity of hexokinase in liver. Increased hexokinase activity was observed in the STZ-induced diabetic rats treated with SAC which would have resulted in the activation of glycolysis, which, in turn, increased the utilization of glucose by restored insulin secretion in the treated rats. The hepatic glucose-6-phosphatase catalyses the terminal step of glucose production and it plays a key role in the maintenance of blood glucose homeostasis. Increased activity of glucose-6-phosphatase in diabetic rats provide hydrogen which binds with NADP+ in the form of NADPH and enhances synthesis of fats from carbohydrates, i.e., lipogenesis (Bopanna et al., 1997) and finally contribute to increased levels of glucose in blood. In the diabetic state, several workers have observed increased activities of glucose-6-phosphatase (Venkateswaran and Pari, 2002). Activation of glucose-6-phosphatase is due to state of insulin deficiency since under normal condition insulin function as a suppressor of glucose-6phosphatase enzymes. Our results demonstrated that hepatic glucose-6-phosphatase activity in diabetic rats was significantly higher than that of normal rats and the oral feeding of SAC and glyclazide markedly lowered its activity. The reduction in enzyme activity corresponded to the decrease in serum glucose as less glucose was being produced and released into the blood stream. Fructose1-6-phosphatase is the important regulatory enzyme of the gluconeogenic pathway (Minnassian and Mitheux, 1994) and it catalyze the rate limiting step of fructose1,6-bisphosphate to fructose-6-phosphate. The activities of fructose1-6-phosphatase are increased in the liver in diabetes. This results in a decrease in the glycolytic flux. Under normal conditions, insulin functions as a suppressor of gluconeogenic enzymes (Baquer et al., 1998). The increased activities of these gluconeogenic enzymes in diabetic rats were decreased to near-normal levels after the administration of SAC and glyclazide. The possible mechanism by which SAC bring about the normalization of enzyme activity may be by potentiation of insulin release from b-cells of the islets of Langerhan’s which might enhance glucose utilization. Glycogen is the primary intracellular storable form of glucose and its levels in various tissues especially skeletal muscle are direct reflection of insulin activity (Garvey, 1992) and it has been previously demonstrated that glycogen deposition from glucose is impaired in diabetic animals (Bollen et al., 1998) in proportion to the severity of insulin deficiency (Gannon and Nuttall, 1997) due to the damage of Langerhans caused by STZ. The previous study (Ikino et al., 1989) showed that tissue glycogen content was reduced drastically in diabetic animals. Treatment of SAC and glyclazide to diabetic rats significantly stimulates the secretion of insulin there by improved glycogen content of liver. The loss of body weight in diabetic control animals may be due to increased catabolism of glycogen in muscle and liver, which may be utilized for energy expenditure instead of being stored. Hepatic glycogen metabolism is controlled by the coordinated action of two enzymes, glycogen synthase and glycogen phosphorylase, both of which are regulated by phosphorylation and allosteric modulators (Ferrer et al., 2003). It has been reported that STZ-induced diabetes pro-
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339
duces partial or total deficiency of insulin that results in decreasing the activity of glycogen synthase and increasing the activity of glycogen phosphorylase (Gayathri and Kannabiran, 2008) leads to decreasing the storage of tissue glycogen (Senthil Kumar et al., 2006). Administration of SAC and glyclazide to diabetic rats restored the level of liver glycogen by decreasing glycogen phosphorylase activity and increasing glycogen synthase activity. In conclusion our result indicates that SAC possess antidiabetic action. The present investigation draws out a sequential metabolic correlation between increased glycolysis and decreased gluconeogenesis and normal glycemia stimulated by SAC which may have been the biochemical mechanism through which glucose homeostasis is regulated.
Acknowledgement The authors thank The Management of KSR College of Arts and Science, Tiruchengode, India and Dr. N. Kannan, Principal of this college for their encouragement and also Management of Sastra University, Thanjavore, India for providing facilities to do animal studies.
R E F E R E N C E S
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McKenna, D. J., Jones, K., Hughes, K., & Humphrey, S. (2002). Botanical medicines, the desk reference for major herbal supplements (2nd ed.). New York, NY: The Haworth Herbal Press. pp. 22–28. Minnassian, C., & Mitheux, G. (1994). Differential time course of liver and kidney glucose-6-phosphatase activity during fasting in rats. Comparative Biochemistry and Physiology – Part B: Biochemistry & Molecular Biology, 109, 99–104. Moriguchi, T., Matsura, H., Kodera, Y., Itakura, Y., Katsuki, H., Saito, H., & Nishiyama, N. (1997). Neurotrophic activity of organosulfur compounds having a thioallyl group on cultured rat hippocampal neurons. Neurochemistry Research, 22, 1449–1452. Murray, R. K., Granner, D. K., Mayes, P. A., & Rodwell, V. W. (2000). Harper’s Biochemistry (25th ed.). Stanford, Connecticut: Appleton & Lange. pp. 610–617. Ong, K. C., & Khoo, H.-E. (2000). Effects of myricetin on glycemia and glycogen metabolism in diabetic rats. Life Sciences, 67, 1695–1705. Palsamy, P., & Subramanian, S. (2008). Resveratrol, a natural phytoalexin, normalizes hyperglycemia in streptozotocinnicotinamide induced experimental diabetic rats. Biomedicine & Pharmacotherapy, 62, 598–605. Papaccio, G., Pisanti, F. A., Latronico, M. V., Ammendola, E., & Galdieri, M. (2000). Multiple low dose and single high dose treatments with streptozotocin do not generate nitric oxide. Journal Cell Biochemistry, 77, 82–91. Pulido, N., Suarez, A., Casanova, B., Romero, R., Rodriguez, E., & Rovira, A. (1997). Glyclazide treatment of streptozotocin diabetic rats restores GLUT4 protein content and basal glucose uptake in skeletal muscle. Metabolism, 46, 10–13. Kaleem, M., Asif, M., Ahmed, Q., & Bano, B. (2006). Antidiabetic and antioxidant activity of Annona squamosa extract in streptozotocin induced diabetic rats. Singapore Medical Journal, 47(8), 670–675. Saravanan, G., Ponmurugan, P., Senthil kumar, G. P., & Rajarajan, T. (2009). Antidiabetic properties of S-allylcysteine, a garlic component on streptozotocin-induced diabetes in rats. Journal of Applied Biomedicine, 7, 151–159. Senthil kumar, G. P., Arulselvan, P., Sathiskumar, D., & Subramanian, S. (2006). Antidiabetic activity of fruits of Terminalia chebula on streptozotocin induced diabetes in rats. Journal of Health Science, 52, 283–291. Venkateswaran, S., & Pari, L. (2002). Effect of Coccinia indica on blood glucose, insulin and hepatic key enzymes in experimental diabetes. Pharmaceutical Biology, 40, 165–170.
1 ( 2 0 0 9 ) 3 3 6 –3 4 0
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jff
Modulatory effect of S-allylcysteine on glucose metabolism in streptozotocin induced diabetic rats Ganapathy Saravanana,b,*, Ponnusamy Ponmurugana,c, Gandhipuram Periasamy Senthilkumarb, Thatchinamoorthi Rajarajand a
Research and Development Centre, Bharathiyar University, Coimbatore 641046, Tamilnadu, India Department of Biochemistry, Centre for Biological science, K.S.R. College of Arts and Science, Thokkavadi, Tiruchengode 637215, Tamil Nadu, India c Department of Biotechnology, K.S.R. College of Technology, Thokkavadi, Tiruchengode 637215, Tamil Nadu, India d Department of Biochemistry, School of Biotechnology, Sastra University, Thanjavore, Tamil Nadu, India b
A R T I C L E I N F O
A B S T R A C T
Article history:
The antihyperglycemic effect of S-allylcysteine (SAC) on normal and streptozotocin (STZ)
Received 14 May 2009
diabetic rats was investigated. Diabetes was induced into male albino Wistar rats by intra-
Received in revised form
peritoneal administration of STZ. The SAC was administered orally (150 mg/kg bw) to nor-
4 August 2009
mal and STZ-diabetic rats for 45 days. The diabetic rats showed an increase in levels of
Accepted 1 September 2009
blood glucose. In addition, diabetic rats showed a significant reduction in the activity of
Available online 23 September 2009
hexokinase, glycogen synthase, liver glycogen and an elevation in the activities of enzymes such as glucose-6-phosphatase, fructose-1,6-bisphosphatase and glycogen phosphorylase.
Keywords:
Treatment with SAC significantly decreased blood glucose and. SAC administration to dia-
S-Allylcysteine
betic rats reversed these enzyme activities in a significant manner. Thus, the results show
Streptozotocin
that SAC possesses an antihyperglycemic activity and provide evidence for its traditional
Diabetes
usage in the control of diabetes. Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Diabetes mellitus (DM) is a group of disorders with different etiologies. It is characterized by derangements in carbohydrate, protein and fat metabolism, caused by complete or relative insufficiency of insulin secretion and/or insulin action (Balkau et al., 2000). According to Food and Agriculture Organization (FAO) projections, the prevalence of diabetes is likely to increase by 35% by the year 2025 (Boyle et al., 2001) and approximately, 140 million people worldwide suffer from diabetes. The disease becomes a real problem of public health in the developing countries, where its prevalence is increasing
steadily. Developing countries like India have today become the diabetic capital of the world with over 20 million diabetics and this number is set to increase to 57 million by 2025 which makes it a country with the highest number of diabetics in the world (King et al., 1998). The control of blood glucose in diabetic patients is achieved mainly by the use of oral hypoglycemic/antihyperglycemic agents and insulin. However, all these treatments have limited efficacy and have been reported to be associated with undesirable side effects (Harrower, 1994). In order to overcome the side effects associated with diabetes, interest has been shifted to the use of alternative medicine.
Abbreviations: SAC, S-allylcysteine; STZ, Streptozotocin * Corresponding author: Present address: PG and Research Department of Biochemistry, Centre for Biological science, K.S.R. College of Arts and Science, Thokkavadi, Tiruchengode 637215, Tamil Nadu, India. E-mail address: [email protected] (G. Saravanan). 1756-4646/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jff.2009.09.001
JOURNAL OF FUNCTIONAL FOODS
Medicinal plants and their bioactive constituents have been extensively used as alternative medicine for better control and management of diabetes mellitus. According to world ethnobotanical information reports, almost 800 plants may possess antidiabetic potential (Alarcon-Aguilara et al., 1998). In the past decade, research has been focused on scientific evaluation of traditional drugs of plant origin and screening of more effective and safe hypoglycemic agents has continued to be an important area. In developing countries 80% of populations are using traditional medicine in primary medical care (Grover and Yadav, 2004). However, a large number of herbs are now being used in the management of DM. India has a wealth of medicinally important plants and ancient herbal treatment methods where traditional alternative medicines are popularly practiced among a large segment of its population. Although several medicinal plants have gained importance for the treatment of diabetes mellitus, many remain to be scientifically investigated. Consumption of garlic (Allium sativum, Liliaceae) and its components has been reported to have many medicinal properties including antidiabetic and anti-lipidemic activities (McKenna et al., 2002). S-allylcysteine is a sulphur-containing amino acid derived from garlic, has been reported to have antioxidant (Herrera-Mundo et al., 2006), anticancer (Chu et al., 2007), and neurotrophic (Moriguchi et al., 1997) activities. The present study was undertaken to determine the effect of S-allylcysteine on diabetes-induced changes in hepatic enzyme activities associated with glucose metabolism with a view to elucidating the probable mechanism by which S-allylcysteine reduces blood glucose.
2.4.
1 ( 20 0 9) 3 3 6–34 0
337
Experimental design
After successful induction of experimental diabetes, the rats were divided into four groups each comprising a minimum of six rats. These were: Group 1, control rats; Group 2, Diabetic control rats; Group 3, Diabetic rats administered with SAC (150 mg/kg bw/rat) in aqueous solution orally for 45 days (Saravanan et al., 2009); and Group 4, Diabetic rats administered with glyclazide (5 mg/kg/bw/rat) in aqueous solution orally for 45 days (Pulido et al., 1997). Body weight and blood glucose level measurements were conducted periodically. At the end of the experimental period, rats were fasted overnight, anaesthetized and sacrificed by cervical decapitation. The blood was collected with or without EDTA (ethylenediaminetetraacetic acid) for plasma or serum separation, respectively.
2.5.
Biochemical assays
Glucose levels were estimated by a commercially available glucose kit based using the glucose oxidase method (Sigma Diagnostics, St. Louis, MO). Liver was immediately dissected, washed in ice-cold saline to remove the blood and homogenised in 0.1 M Tris–HCl buffer, pH 7.4. The supernatant was used for enzyme activity assays. Hexokinase, glucose-6-phosphatase and fructose-1,6-bisphosphatase were assayed by the method of Lapeir and Rodnick (2001), Koida and Oda (1959) and Gancedo and Gancedo (1971), respectively. Glycogen synthase and phosphorylase activities were assayed by the method of Leloir and Goldenber (1962) and Cornblath et al. (1963), respectively. Liver glycogen was assayed by the method of Ong and Khoo (2000).
2.
Materials and methods
2.6.
2.1.
Animals
All the grouped data were statistically evaluated with SPSS/ 10.0 software. Hypothesis testing methods included one way analysis of variance (ANOVA) followed by least significant difference (LSD) test; p value of less than 0.05 were considered to indicate statistical significance. All the results were expressed as the mean ± S.D. for six animals in each group.
Male Wister rats of body weight 150–180 g were obtained from Nandha College of Pharmacy, Erode, India. They were kept in the Animal House, Sastra University, Thanjavore, India, on a standard pellet diet (AMRUT, Pune, India) and water ad libitum. The protocol of this study was approved by the Institutional Ethical Committee of Sastra University, Thanjavore, India.
2.2.
Chemicals
SAC (99%) was purchased from LGC Prochem, Bangalore, India. Streptozotocin was purchased from Himedia, Bangalore, India. All other chemicals used were of analytical grade.
2.3.
Induction of diabetes
Diabetes was induced (Kaleem et al., 2006) in overnight fasted adult Wistar strain albino male rats weighing 150–180 g by a single intraperitoneal injection of 55 mg/kg streptozotocin. Streptozotocin (55 mg/kg) was dissolved in 0.1 M citrate buffer (pH 4.5). Hyperglycemia was confirmed by the elevated glucose levels (Above 250 mg/dl) in plasma, determined at 72 h and then on day 7 after injection.
3.
Statistical analysis
Results
Table 1 shows the glucose level of control and experimental group of rats. The blood glucose level in the control rats was significantly increased in diabetic rats. Treatment with SAC as well as glyclazide to diabetic rats elicited significant (p < 0.05) decreases in blood glucose level when compared with diabetic control rats. Table 2 summarizes the levels of hexokinase, glucose-6phosphatase and fructose-6-phosphatase in the control and experimental groups of rats. A significant decrease in hepatic hexokinase level and concomitant increase in glucose-6phosphatase and fructose-6-phosphatase level was observed in STZ-diabetic rats and it was normalized after treatment of SAC and glyclazide. Table 3 shows the hepatic glycogen content, and in the activity of glycogen synthase and glycogen phosphorylase in control and experimental groups of rats. There was a signifi-
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Table 1 – Effects of SAC on blood glucose and urine sugar in control and experimental animals. Groups
Blood glucose (mg/dl)
Urine sugar
89.15 ± 1.23 276.41 ± 12.63a,* 96.34 ± 1.47b,* 93.68 ± 1.68b,*
Nil +++ Nil Nil
Control Diabetic control Diabetic + SAC (150 mg/kg) Diabetic + glyclazide Values are mean ± S.D., n = 6. Significantly different from control. b Significantly different from diabetic control. * p < 0.05. a
Table 2 – Effects of SAC on hexokinase, glucose-6-phosphatase and fructose-1,6-bisphosphatase in control and experimental animals. Groups Control Diabetic Diabetic + SAC (150 mg/kg) Diabetic + glyclazide
Hexokinase
Glucose-6-phosphatase
265.41 ± 11.93 136.73 ± 9.42 a,* 252.68 ± 10.12 b,* 256.54 ± 9.63b,*
965.13 ± 46.48 1600.14 ± 120.95 a,* 1042.10 ± 46.88 b,* 1012.22 ± 48.54b,*
Fructose-1,6-bisphosphatase 465.13 ± 22.45 750.65 ± 63.53 a,* 660.31 ± 24.90 b,* 575.16 ± 26.34b,*
Values are mean ± S.D., n = 6. Hexokinase: lmol of glucose phosphorylated/min/mg of protein, glucose-6-phosphatase: lmol of Pi liberated/min/mg of protein, fructose-1,6bisphosphatase: lmol of phosphate liberated/min/mg of protein. a Significantly different from control. b Significantly different from diabetic control. * p < 0.05.
Table 3 – Effects of SAC on glycogen, glycogen synthase and glycogen phosphorylase in control and experimental animals. Groups Control Diabetic Diabetic + SAC (150 mg/kg) Diabetic + glyclazide
Glycogen (mg/g wet tissue) 56.15 ± 2.98 22.40 ± 1.15a,* 50.65 ± 1.95b,* 52.32 ± 1.68b,*
Glycogen synthase 848.54 ± 72.34 462.36 ± 17.98a,* 810.63 ± 66.13b,* 823.95 ± 68.21b,*
Glycogen phosphorylase 610.21 ± 23.15 845.32 ± 33.49a,* 718.10 ± 26.10b,* 712.48 ± 22.14b,*
Values are mean ± S.D., n = 6. Glycogen synthase: lmol of uridine diphosphate formed/h/mg protein glycogen phosphorylase: lmol of phosphate liberated/h/mg protein. a Significantly different from control. b Significantly different from diabetic control. * p < 0.05.
cant reduction in hepatic glycogen content and activity of glycogen synthase and concomitant increase in the activity of glycogen phosphorylase in STZ-diabetic rats when compared with control rats. Oral treatment of SAC and glyclazide tended to bring glycogen content, glycogen synthase and glycogen phosphorylase towards near-normal levels.
4.
Discussion
Streptozotocin is well known for its selective pancreatic islet b-cell cytotoxicity and has been extensively used to induce diabetes mellitus in animals. It interferes with cellular metabolic oxidative mechanisms (Papaccio et al., 2000). Intraperitoneal administration of streptozotocin effectively induced diabetes in normal rats, as observed by hyperglycemia when compared with normal rats. Persistent hyperglycemia, the common characteristic of diabetes can cause most diabetic complications and it is normalized by the action of insulin
(Gayathri and Kannabiran, 2008). In the present study, it was observed that oral administration of SAC could reverse the above mentioned diabetic effect, possibly due to an insulinlike effect of SAC on peripheral tissues, either by promoting glucose uptake and metabolism, or by inhibiting hepatic gluconeogenesis. A number of compounds have also been shown to exert hypoglycemic activity through stimulation of insulin release (Palsamy and Subramanian, 2008). The hypoglycemic potency of SAC was comparable glyclazide, a standard hypoglycemic drug. Glyclazide has long been used to treat diabetes and is known to act by stimulating insulin secretion through action on the pancreatic b-cells (Jeong-Kwon Park et al., 2008). Hexokinase is an insulin-dependent and insulin-sensitive enzyme and are almost completely inhibited or inactivated in diabetic rat liver in the absence of insulin (Gupta et al., 1997). Decreased enzymatic activity of hexokinase has also been reported in diabetic animals, resulting in depletion of liver and muscle glycogen (Murray et al., 2000) . Administration
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of SAC and glyclazide to STZ treated rats resulted in an increased activity of hexokinase in liver. Increased hexokinase activity was observed in the STZ-induced diabetic rats treated with SAC which would have resulted in the activation of glycolysis, which, in turn, increased the utilization of glucose by restored insulin secretion in the treated rats. The hepatic glucose-6-phosphatase catalyses the terminal step of glucose production and it plays a key role in the maintenance of blood glucose homeostasis. Increased activity of glucose-6-phosphatase in diabetic rats provide hydrogen which binds with NADP+ in the form of NADPH and enhances synthesis of fats from carbohydrates, i.e., lipogenesis (Bopanna et al., 1997) and finally contribute to increased levels of glucose in blood. In the diabetic state, several workers have observed increased activities of glucose-6-phosphatase (Venkateswaran and Pari, 2002). Activation of glucose-6-phosphatase is due to state of insulin deficiency since under normal condition insulin function as a suppressor of glucose-6phosphatase enzymes. Our results demonstrated that hepatic glucose-6-phosphatase activity in diabetic rats was significantly higher than that of normal rats and the oral feeding of SAC and glyclazide markedly lowered its activity. The reduction in enzyme activity corresponded to the decrease in serum glucose as less glucose was being produced and released into the blood stream. Fructose1-6-phosphatase is the important regulatory enzyme of the gluconeogenic pathway (Minnassian and Mitheux, 1994) and it catalyze the rate limiting step of fructose1,6-bisphosphate to fructose-6-phosphate. The activities of fructose1-6-phosphatase are increased in the liver in diabetes. This results in a decrease in the glycolytic flux. Under normal conditions, insulin functions as a suppressor of gluconeogenic enzymes (Baquer et al., 1998). The increased activities of these gluconeogenic enzymes in diabetic rats were decreased to near-normal levels after the administration of SAC and glyclazide. The possible mechanism by which SAC bring about the normalization of enzyme activity may be by potentiation of insulin release from b-cells of the islets of Langerhan’s which might enhance glucose utilization. Glycogen is the primary intracellular storable form of glucose and its levels in various tissues especially skeletal muscle are direct reflection of insulin activity (Garvey, 1992) and it has been previously demonstrated that glycogen deposition from glucose is impaired in diabetic animals (Bollen et al., 1998) in proportion to the severity of insulin deficiency (Gannon and Nuttall, 1997) due to the damage of Langerhans caused by STZ. The previous study (Ikino et al., 1989) showed that tissue glycogen content was reduced drastically in diabetic animals. Treatment of SAC and glyclazide to diabetic rats significantly stimulates the secretion of insulin there by improved glycogen content of liver. The loss of body weight in diabetic control animals may be due to increased catabolism of glycogen in muscle and liver, which may be utilized for energy expenditure instead of being stored. Hepatic glycogen metabolism is controlled by the coordinated action of two enzymes, glycogen synthase and glycogen phosphorylase, both of which are regulated by phosphorylation and allosteric modulators (Ferrer et al., 2003). It has been reported that STZ-induced diabetes pro-
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duces partial or total deficiency of insulin that results in decreasing the activity of glycogen synthase and increasing the activity of glycogen phosphorylase (Gayathri and Kannabiran, 2008) leads to decreasing the storage of tissue glycogen (Senthil Kumar et al., 2006). Administration of SAC and glyclazide to diabetic rats restored the level of liver glycogen by decreasing glycogen phosphorylase activity and increasing glycogen synthase activity. In conclusion our result indicates that SAC possess antidiabetic action. The present investigation draws out a sequential metabolic correlation between increased glycolysis and decreased gluconeogenesis and normal glycemia stimulated by SAC which may have been the biochemical mechanism through which glucose homeostasis is regulated.
Acknowledgement The authors thank The Management of KSR College of Arts and Science, Tiruchengode, India and Dr. N. Kannan, Principal of this college for their encouragement and also Management of Sastra University, Thanjavore, India for providing facilities to do animal studies.
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