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Effect of tangeretin, a polymethoxylated flavone on glucose metabolism in streptozotocin-induced diabetic rats
Phytomedicine 21 (2014) 793–799
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Effect of tangeretin, a polymethoxylated flavone on glucose metabolism in streptozotocin-induced diabetic rats Ramalingam Sundaram a , Palanivelu Shanthi b , Panchanatham Sachdanandam a,∗ a Department of Medical Biochemistry, Dr. ALM P-G, Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, Tamil Nadu 600113, India b Department of Pathology, Dr. ALM P-G, Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, Tamil Nadu 600113, India
a r t i c l e
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Article history: Received 29 August 2013 Received in revised form 11 November 2013 Accepted 26 January 2014 Keywords: Tangeretin Diabetes Carbohydrate metabolism Cardiovascular disease
a b s t r a c t The present study was designed to evaluate the antihyperglycemic potential of tangeretin on the activities of key enzymes of carbohydrate and glycogen metabolism in control and streptozotocin induced diabetic rats. The daily oral administration of tangeretin (100 mg/kg body weight) to diabetic rats for 30 days resulted in a significant reduction in the levels of plasma glucose, glycosylated hemoglobin (HbA1c) and increase in the levels of insulin and hemoglobin. The altered activities of the key enzymes of carbohydrate metabolism such as hexokinase, pyruvate kinase, lactate dehydrogenase, glucose-6phosphatase, fructose-1,6-bisphosphatase, glucose-6-phosphate dehydrogenase, glycogen synthase and glycogen phosphorylase in liver of diabetic rats were significantly reverted to near normal levels by the administration of tangeretin. Further, tangeretin administration to diabetic rats improved hepatic glycogen content suggesting the antihyperglycemic potential of tangeretin in diabetic rats. The effect produced by tangeretin on various parameters was comparable to that of glibenclamide – a standard oral hypoglycemic drug. Thus, these results show that tangeretin modulates the activities of hepatic enzymes via enhanced secretion of insulin and decreases the blood glucose in streptozotocin induced diabetic rats by its antioxidant potential. © 2014 Elsevier GmbH. All rights reserved.
Introduction Diabetes mellitus (DM) is a chronic metabolic disease with the highest rates of prevalence and mortality worldwide that is caused by an absolute or relative lack of insulin and or reduced insulin activity, which results in hyperglycemy and abnormalities in carbohydrate, protein, and fat metabolism (Kamtchouing et al. 2006; Fatima et al. 2010). The World Health Organization (WHO) estimates that more than 220 million people worldwide have diabetes, and this number is liable to double by 2030 (WHO 2009). The chronic hyperglycaemia of diabetes is associated with damage, dysfunction and failure of various organs such as kidneys, retina, heart, liver, peripheral and central nervous system (Shanmugam et al. 2011). Recently, much attention has been focused on screening of products from natural sources, such as flavonoids, that may be beneficial for reducing the risk for metabolic syndrome (Sabu et al. 2002; Jung
∗ Corresponding author. Tel.: +91 9884855505. E-mail addresses: [email protected] (P. Shanthi), [email protected] (P. Sachdanandam). 0944-7113/$ – see front matter © 2014 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2014.01.007
et al. 2004), because products from plant sources are usually considered to be less toxic and have fewer side effects than products from synthetic sources. Tangeretin is a polymethoxylated flavones abundant in the citrus fruit rinds, including mandarin orange, Poncirus trifoliate Raf. Tangeretin plays an important role in preventing various diseases including cancer, oxidative stress, and inflammation (Yoon et al. 2011; Xu et al. 2008). In addition to its anti-oxidant effects, tangeretin has been reported to inhibit the growth of hepatocytes both in vitro and in vivo via inhibition of mTOR/p70S6 kinase (Cheng et al. 2011). Regarding tangeretin mediated effects on neuronal disease, a number of studies have shown that tangeretin reduces dopaminergic neurotoxin-induced neuronal injury and prevents tunicamycin-induced cell death in mice through an increase in glucose-regulated protein (GRP)78 and heme oxygenase (HO)-1 expression in renal tubular epithelium (Takano et al., 2007). A recent study investigating the anti-inflammatory effects of citrus fruit peels showed that tangeretin had a beneficial effect on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cells, which may provide protection against disease resulting from excessive NO production (Choi et al. 2007).
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OCH 3 O
Experimental design
H3CO H3CO
O OCH 3
OCH 3
Fig. 1. Tangeretin.
Considering the wide variety of pharmacological action of tangeretin, the present study was undertaken to explore the tangeretin on key hepatic enzyme in streptozotocin induced diabetic rats. The effect of tangeretin was compared with conventional antidiabetic agent glibenclamide. The chemical structure of tangeretin was given in Fig. 1.
Materials and methods Sources of chemicals All fine chemicals including streptozotocin were purchased from Sigma Chemical Company (St. Louis, MO, USA). All other chemicals used were of good quality and analytical grade and obtained from Himedia, Mumbai, India.
Animals Male albino Wistar rats weighing 180–200 g m body weight were procured from the Central Animal House Facility, University of Madras, Taramani Campus, Chennai, Tamil Nadu, India. They were maintained at an ambient temperature of 25 ± 2 ◦ C and 12/12 h of light/dark cycle. Animals were given standard commercial rat chow and water ad libitum and housed under standard environmental conditions throughout the study. The experiments were conducted according to the ethical norms approved by the Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines.
Experimental induction of diabetes Diabetes was induced in overnight fasted experimental rats by single intraperitoneal injection of streptozotocin (60 mg/kg bodyweight) dissolved in freshly prepared 0.2 ml of 0.1 mol/l citrate buffer, pH 4.5. Streptozotocin injected animals were allowed to drink 20% glucose solution overnight to overcome the initial druginduced hypoglycemic mortality. Control rats were injected with the vehicle (0.2 ml of 0.1 mol/l citrate buffer, pH 4.5) alone. After 96 h, plasma glucose was determined and those rats with fasting blood glucose greater than 300 mg/dl were used in the present study. In diabetic rats, the treatment was started six weeks after the onset of diabetes because, in many studies, the duration of diabetes required to induce cardiac dysfunction in experimental rats has been found to be six weeks (Paulson 1997). Oral glucose tolerance test (OGTT) was performed according to the method of Du Vigneaud and Karr (1925). After overnight fasting, ‘0 minute blood sample (0.2 ml) was taken from control and experimental rats. Without delay, a glucose solution (2 g/kg body weight) was administered by oral gavage. Blood samples were taken at 30, 60, 90 and 120 min after glucose administration. Blood samples were collected with potassium oxalate and sodium fluoride and glucose levels were determined by the kit method of Trinder (1969).
The animals were randomly divided into seven groups of six animals in each (30 diabetic surviving and 12 normal). Group I: Control animals (normal healthy control rats received intra gastrically 0.5 ml of 0.9% saline for 30 days. Group II: Drug control (normal healthy control rats received intra gastrically tangeretin (100 mg/kg b.w.) dissolved in 0.5 ml of 0.9% saline for 30 days. Group III: Diabetic control rats. Group IV: Diabetic rats received intra gastrically tangeretin (25 mg/kg b.w) dissolved in 0.5 ml of 0.9% saline for 30 days. Group V: Diabetic rats received intra gastrically tangeretin (50 mg/kg b.w) dissolved in 0.5 ml of 0.9% saline for 30 days. Group VI: Diabetic rats received intra gastrically tangeretin (100 mg/kg b.w) dissolved in 0.5 ml of 0.9% saline for 30 days. Group VII: Diabetic rats received intra gastrically glibenclamide (5 mg/kg b.w) dissolved in 0.5 ml of 0.9% saline for 30 days. At the end of the treatment period (75 days), the rats were fasted overnight, anaesthetized and sacrificed by cervical decapitation. The blood was collected with or without EDTA for plasma or serum separation, respectively. The liver tissue was dissected out, washed in ice-cold saline, and weighed. Tissue was minced and homogenized (10%, w/v) with 0.1 M Tris–HCl buffer (pH 7.4) and centrifuged (3000 × g for 10 min). The resulting supernatant was used for enzyme assays. Body weights of all the animals were recorded prior to the treatment and sacrifice. Food and water intake of all groups of animals were monitored on a daily basis for 30 days at a fixed time. Fixed amount of rat chow and fluid was given to each rat and replenished the next day. Biochemical analysis The level of plasma glucose was estimated spectrophotometrically using commercial diagnostic kit (Agappe Diagnostics Pvt. Ltd., Kerala, India) Trinder (1969). Plasma insulin level was assayed by enzyme linked immunosorbent assay kit (ELISA) (Boehringer Mannheim kit). Hemoglobin and HbA1C was estimated by diagnostic kit (Agappe Diagnostic Pvt. Ltd., India) Bisse and Abragam, (1985). The estimation of protein was carried out by the method of Lowry et al. (1951). Hepatic hexokinase activity was assayed by the method of Brandstrup et al. (1957). Glucose-6-phosphate dehydrogenase was assayed by the method of Ellis and Kirkman (1961). Glucose-6-phosphatase was assayed by the method of Koide and Oda (1959). Fructose-1,6-bisphosphatase activity was measured by the method of Gancedo and Gancedo (1971), pyruvate kinase activity was estimated by the method of Pogson and Denton (1967), lactate dehydrogenase activity was estimated by the method of King (1965), glycogen synthase activity was estimated by the method of Leloir and Goldemberg (1962), glycogen phosphorylase activity was estimated by the method of Cornblath et al. (1963). Another portion of wet liver glycogen content was estimated by the method of Morales et al. (1973). Histopathological studies Pancreatic tissues were harvested from the sacrificed animals and were fixed in 10% neutral buffered formalin solution, dehydrated in ethanol and embedded in paraffin. Sections of 5 m thickness were prepared using a rotary microtome and stained with hematoxylin and eosin dye and mounted in neutral deparaffinated xylene medium for microscopic observations.
R. Sundaram et al. / Phytomedicine 21 (2014) 793–799 Table 1 Dose-dependent effects of tangeretin on changes in plasma glucose and insulin in normal and diabetic rats. Group
Glucose (mg/dl)
Control Normal + tangeretin (100 mg/kg b.w.) Diabetic control Diabetic + tangeretin (25 mg/kg b.w.) Diabetic + tangeretin (50 mg/kg b.w) Diabetic + tangeretin (100 mg/kg b.w.) Diabetic + glibenclamide (5 mg/kg)
90.33 87.0 323.33 280.93 220.37 133.16 128.33
± ± ± ± ± ± ±
8.64 6.78 24.42b 10.47c 14.16d 10.04e 7.20f
Insulin (U/ml) 17.73 18.76 6.26 8.56 11.25 15.98 16.35
± ± ± ± ± ± ±
1.24 1.52 0.53b 0.51c 0.73d 1.49e 1.23f
Values are given as mean ± S.D. for six animals in each group (n = 6), Values are considered significantly different at p < 0.05 with post hoc LSD test. a Control vs drug control (tangeretin alone treated rat). b Control rat vs diabetic rat. c Diabetic rat vs tangeretin 25 mg/kg. d Diabetic rat vs tangeretin 50 mg/kg. e Diabetic rat vs tangeretin 100 mg/kg. f Tangeretin (100 mg/kg) treated diabetic rat vs glibenclamide treated diabetic rats (5 mg/kg).
Statistical analysis The results are expressed as mean ± standard deviation (S.D.). Differences between groups were assessed by ANOVA using the SPSS software package for Windows. Post hoc testing was performed for inter-group comparisons using the least significance difference (L.S.D.) p-values < 0.05 were considered as significantly altered. Results Estimation of plasma glucose, insulin and glucose tolerance Table 1 shows the levels of plasma glucose, insulin in normal and experimental rats. A significant increase in plasma glucose and a decrease in insulin levels were observed in diabetic rats. However, administration of tangeretin to diabetic rats significantly decreased the blood glucose levels to near normal. There was no significant change in normal rats treated with tangeretin. Results of OGTT conducted on control and different experimental groups are shown in Fig. 2. After the oral dose of glucose in normal control rats the blood glucose reached the fasting levels at 2 h. In diabetic control rats, the blood glucose levels remained higher even after 2 h. Treatment with tangeretin showed a significant decrease in blood glucose levels. The maximum glucose lowering effect of tangeretin was observed at a dose of 100 mg/kg body weight than the other two doses. Therefore, further studies were carried out with this dose. Determination of hemoglobin and HbA1C The levels of hemoglobin (Hb) and glycosyated hemoglobin (HbA1C) in control and experimental animals were depicted in Table 2. Decreased Hb and an increase in HbA1C were observed in diabetic rats. These values improved toward near normal on treatment with tangeretin and glibenclamide. However, no significant variations were found in the control rats treated with tangeretin alone for a period of 30 days. Changes in body weight, organ weight, food and water intake The changes in the body weight, liver weight, food and water intake of control and experimental animals were represented in Table 3. The baseline body weight at the beginning of the study was similar in all groups. At the end of the experimental period, the body weight and liver weight was significantly decreased
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in diabetic rats when compared to control rats. However, oral administration of tangeretin and glibenclamide to diabetic rats reversed the body weight and liver weight changes to near normal. Normal rats treated with tangeretin showed no significant changes in the body weight and liver weight. Diabetic animals also showed signs of polyuria, polydipsia and polyphagia. The food and water intake increased in diabetic rats compared with control rats (Table 3). Administration of tangeretin and glibenclamide significantly decreased the food and water intake in diabetic rats. Activities of carbohydrate metabolic enzymes Table 4 portrays the changes in the activities of carbohydrate metabolizing enzymes in the liver of control and experimental rats. The activities of hexokinase, pyruvate kinase and glucose-6phosphate dehydrogenase were significantly decreased whereas the activities of lactate dehydrogenase. Glucose-6-phosphatase and fructose-1,6-biphosphatase were significantly increased in diabeticrats when compared to normal control rats. However, upon treatment with tangeretin and glibenclamide to diabetic rats reversed the activities of these hepatic key enzymes to near normal. Glycogen content and activities of glycogen metabolic enzymes Liver glycogen metabolism of control and experimental animals were shown in Table 5. A significant decline in the glycogen level as well as in the glycogen synthase activity and a concomitant increase in the activity of glycogen phosphorylase were noted in the liver of diabetic rats. Oral treatment with tangeretin and glibenclamide to diabetic groups of rats reinstated the level of glycogen and the activities of glycogen synthase and glycogen phosphorylase to near normal when compared to control rats. Histological examination of pancreas Fig. 3 represents the microphotographs of H&E staining of pancreatic tissues of normal control and experimental rats. Histopathological examination showed that streptozotocin and high fat diet administration elicited severe injury in pancreatic ß-cells, such as a decrease of islet cells’ number and vascular degenerative changes in the islets (Fig. 3C). The pancreas of the control and drug control rat showed normal islets and well organized dark beta cells (Fig. 3A and B). Both the tangeretin and glibenclamide treated diabetic rats showed the regenerating islets an increase in the islets as compared with the diabetic control group (Fig. 3D and E). Discussion Streptozotocin (STZ)-induced hyperglycemia in animals is considered to be a good model for the preliminary screening of agents active against diabetes and is widely used (Ivorra et al. 1989). In experiment with many animal species, streptozotocin produces permanent diabetes that impersonates the pathological status of human diabetes (Larson et al., 2002). Therefore, streptozotocin induced diabetes is reproducible, convenient and can produce diabetes of graded severity suitable for experimental diabetes (Srinivasan and Ramarao 2007). The mechanism by which streptozotocin brings about its diabetic state includes selective destruction of pancreatic beta cells which make cells less active and leading to poor sensitivity of insulin for glucose uptake by tissues which causes hyperglycemia (Jacot and Assal 1989; Marles and Farnsworth 1995). The persistent supra-physiological level of glucose non-enzymatically reacts with hemoglobin to form increased glycosylated hemoglobin which is a standard biochemical marker for the diagnosis of ambient glycemia during a period
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Fig. 2. Oral glucose tolerance test.
Table 2 Effect of tangeretin on the levels of Hb and HbA1c in normal and diabetic rats. Parameters
Normal control
Normal + tangeretin (100 mg/kg b.w.)
Diabetic control
Diabetic + tangeretin (100 mg/kg b.w.)
Diabetic + glibenclamide (5 mg/kg bw)
Hb (g/dl) HbA1c (mg/g of Hb)
14.38 ± 1.04 4.75 ± 0.33
13.96 ± 1.07 4.22 ± 0.31
7.44 ± 0.54b 13.41 ± 1.05b
12.01 ± 0.93c 6.18 ± 0.58c
12.66 ± 1.21 5.83 ± 0.41
Values are given as mean ± S.D. for six animals in each group (n = 6), Values are considered significantly different at p < 0.05 with post hoc LSD test. a Control vs Drug control (Tangeretin alone treated rats). b Control rats vs Diabetic rats. c Diabetic rats vs Tangeretin treated diabetic rats. d Tangeretin treated diabetic rats vs Glibenclamide.
of 3-month (Alberti et al. 1982). The oral administration of tangeretin and glibenclamide to diabetic rats for 30 days significantly reversed plasma insulin, blood glucose, glycosylated hemoglobin and hemoglobin. Based on the results obtained from this study, it can be concluded that the elevated levels of pancreatic insulin may have enhanced the glucose utilization by peripheral tissues of diabetic rats either by promoting glucose uptake and metabolism, or by inhibiting hepatic gluconeogenesis and decreased blood glucose levels. In previous investigations the diabetic animals displayed the following characteristics polyuria, increased water intake, dehydration, weight loss and muscle wasting, excessive hair loss
and scaling, diarrhea, cataracts and increased food intake (Pari and Rajarajeswari 2009). Decrease in body weight of diabetic rats is due to catabolism of fats and proteins. Due to insulin deficiency the protein content is decreased in muscular tissue by proteolysis (SubashBabu et al. 2007). On oral administration of tangeretin and glibenclamide prevented the loss of body weight, excess of food and fluid intake in diabetic rats by controlling the hyperglycemia. Liver is a general metabolic organ that plays a pivotal role in glycolysis and gluconeogenesis. In the present study, a marked decrease in liver weight was observed in diabetic rats. This could be due to an increased breakdown of glycogen, protein degradation and increased gluconeogenesis. This result corroborates with
Table 3 Body weight, food and fluid intake in normal and diabetic rats before and after oral treatment with tangeretin. Parameters
Normal control
Body weight initial (g) Body weight final (g) Liver weight (g) Food intake after (g/rat/day) Fluid intake before (ml/rat/day) Fluid intake after (ml/rat/day)
244.6 467.1 12.07 14.58 72.16 70.66
± ± ± ± ± ±
10.57 12.08 1.15 1.49 5.49 5.35
Normal + tangeretin (100 mg/kg b.w.) 240.8 464 12.42 14.13 71.5 68.83
± ± ± ± ± ±
9.70 13.14 0.92 0.82 6.22 4.87
Diabetic control 240.6 224.6 7.55 65.03 128.1 137.5
± ± ± ± ± ±
8.75 8.28b 0.70b 5.69b 6.79 8.24b
Diabetic + tangeretin (100 mg/kg b.w.) 242.6 442.5 10.71 27.36 114.3 88.66
± ± ± ± ± ±
6.18 16.35c 0.76c 1.88c 8.64 5.20c
Diabetic + glibenclamide (5 mg/kg bw) 246.3 447.3 11.01 26.10 112 85
Values are given as mean ± S.D. for six animals in each group (n = 6). Values are considered significantly different at p < 0.05 with post hoc LSD test. a Control vs Drug control (Tangeretin alone treated rats). b Control rats vs Diabetic rats. c Diabetic rats vs Tangeretin treated diabetic rats. d Tangeretin treated diabetic rats vs Glibenclamide.
± ± ± ± ± ±
8.77 15.25 0.73 1.64 7.66 6.03
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Table 4 Effect of tangeretin on the activities of carbohydrate metabolizing enzymes in the liver of normal and diabetic rats. Parameters
Normal control
Normal + tangeretin (100 mg/kg b.w.)
Diabetic control
Diabetic + tangeretin (100 mg/kg b.w.)
Diabetic + glibenclamide (5 mg/kg bw)
Hexokinase Lactate dehydrogenase Pyruvate kinase Glucose 6 phosphatase Fructose 1,6 bisphopatase Glucose 6 phophatase
254.6 ± 14.5 239 ± 13.28 8.97 ± 0.80 1040 ± 15.79 494.3 ± 21.62 548.5 ± 27.48
257.1 ± 10.70 235.5 ± 13.32 9.44 ± 0.76 1037 ± 16.14 492.1 ± 23.43 551.6 ± 27.78
126.5 ± 11.2b 438.3 ± 18.98b 3.23 ± 0.31b 1925 ± 31.10b 805.6 ± 30.91b 266.5 ± 15.93b
205.5 ± 12.9c 268.8 ± 10.20c 7.42 ± 0.70c 1141 ± 28.88c 518.8 ± 25.95c 496.1 ± 11.73c
208.8 ± 8.56 265.6 ± 9.33 7.99 ± 0.76 1138 ± 27.78 515.1 ± 26.07 502.1 ± 11.12
Hexokinase – moles glucose-6-phosphatase formed in hour/mg of protein, lactate dehydrogenase – moles of pyruvate formed/hour/mg of protein, pyruvate kinase – moles of pyruvate formed in minute/mg of protein, glucose 6 phosphatase, fructose 1,6 bisphopatase – moles phosphate liberated in h/mg of protein, glucose-6-phophate dehydrogenase – Units/min per mg of protein. Values are given as mean ± S.D. for six animals in each group (n = 6). Values are considered significantly different at p < 0.05 with post hoc LSD test. a Control vs Drug control (Tangeretin alone treated rats). b Control rats vs Diabetic rats. c Diabetic rats vs Tangeretin treated diabetic rats. d Tangeretin treated diabetic rats vs Glibenclamide.
previous report (Anand et al. 2010). Tangeretin and glibenclamide, treatment has elevated the liver weight in diabetic rats by reversing gluconeogenesis. Further, it was confirmed by elevated levels of glycogen synthase and declined the levels of glycogen phospharylase in the liver of diabetic treated rats which in turn increase the glycogen content in liver. Elevated endogenous glucose production is a common abnormality associated with diabetes that, in concurrence with deprived pancreatic function and reduced glucose clearance, contributes to the hyperglycemia characteristic of the disease, diabetes (Wajngot et al. 2001). Insulin regulates the metabolism by modulating the uptake and utilization of glucose in target organs such as liver, kidney, skeletal muscle and adipose tissue by controlling the activities of numerous metabolic enzymes. Hexokinase is insulin dependent and one of the key enzymes of carbohydrate metabolism which involve the phosphorylation of glucose into glucose-6-phosphate and its level was significantly decreased in the liver of diabetic rats, this may be the reason for the diminished consumption of glucose in the system and increased blood sugar levels due to lack of insulin and loss of insulin receptor (Roden and Bernroide 2003). Administration of tangeretin and glibenclamide to diabetic rats significantly reduces the levels of plasma glucose which activates hexokinase and increase glycolysis and utilization of glucose for energy production via enhanced secretion of insulin from remnant pancreatic  cells. Liver-pyruvate kinase activity decreases as the result of diabetes and increases by the administration of insulin to diabetic rats (Yamada and Noguchi 1999). The altered activity during diabetic conditions could be expected to diminish the metabolism of glucose and ATP production. Hence, the observed decline in the activity of PK in the liver of diabetic rats promptly responsible for the reduced glycolysis and amplified gluconeogenesis signifying that these two pathways are altered in diabetes (Taylor and Agius 1988; Kavanagh et al. 2004). The treatment of tangeretin and glibenclamide to diabetic rats showed a notable increase in plasma
insulin that induces a decrease in ATP, a known allosteric inhibitor of PK, thereby increases the PK activity to near normalcy. Lactate dehydrogenase (LDH) is a terminal glycolytic enzyme that plays an indispensable role in the interconversion of pyruvate to lactate to yield energy under anaerobic conditions and the reaction occurs in both cytosolic and mitochondrial compartments (Ainscow et al. 2000). This enzyme is a tetramer, which can be composed of two different kinds of subunits: M (muscle type) and H (heart type) and the biosynthesis of each of these subunits is apparently controlled by separate genes. LDH activity is found to be altered by insulin, glucose, NADH, as well as increases in mitochondrial membrane potential, cytosolic free ATP and cytosolic free Ca2+ (Shulman 2000). The decreased activity of LDH in tissues could be important to ensure that a high proportion of both pyruvate and NADH, supplied by glycolysis, is subsequently oxidized by mitochondria. Indeed, elevated LDH levels observed in the experimental diabetic animals’ are associated with impaired glucose-stimulated insulin secretion (Pari and Saravanan 2005). Thus, increased activity of LDH interferes with normal glucose metabolism and insulin secretion in the  cells of pancreas and it may therefore be directly responsible for insulin secretory defects in diabetes. However, treatment with tangeretin and glibenclamide to diabetic rats reverted the LDH activity to near normalcy most probably by regulating the proportion of pyruvate and NADH thereby promoting the mitochondrial oxidation of (pyruvate) glucose. Glucose-6-phosphatase and fructose-1,6-bisphosphatase are the important enzymes in regulating of gluconeogenic pathway. The activities of glucose-6-phosphatase and fructose-1,6bisphosphatase were increased in the liver of diabetic rats (Pederson et al. 2005). The activities of the two enzymes may be due to the increased synthesis of enzymes contributing to the increased glucose production during diabetes by the liver (Mitra et al. 1995). Tangeretin inhibits gluconeogensis by inhibiting the activity of glucose-6-phosphatase and fructose-1,6-bisphosphatase. Oral
Table 5 Effect of tangeretin on the levels of glycogen content and activities of glycogen synthase and glycogen phosphorylase in liver tissues of normal and diabetic rats. Parameters
Normal control
Normal + tangeretin (100 mg/kg b.w.)
Diabetic control
Diabetic + tangeretin (100 mg/kg b.w.)
Diabetic + glibenclamide (5 mg/kg bw)
Liver glycogen (mg/g tissue) Glycogen synthase ( moles of UDP formed/h/mg protein) Glycogen phosphorylase ( moles Pi liberated/h/mg protein)
56.34 ± 5.11 860.5 ± 11.7
57.02 ± 5.30 862.5 ± 11.92
15.77 ± 1.45b 547.1 ± 18.25b
48.05 ± 4.14c 802.1 ± 16.24c
48.65 ± 4.30 805.8 ± 15.8
646.5 ± 12.7
648.8 ± 12.3
886.6 ± 23.59b
731.5 ± 17.32c
728.3 ± 16.6
Values are given as mean ± S.D. for six animals in each group (n = 6). Values are considered significantly different at p < 0.05 with post hoc LSD test. Control vs Drug control (Tangeretin alone treated rats). b Control rats vs Diabetic rats. c Diabetic rats vs Tangeretin treated diabetic rats. d Tangeretin treated diabetic rats vs Glibenclamide. a
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Fig. 3. Histopathological changes in pancreas of control and diabetic rats. All the sections are in H&E 40X. (A) Normal control, (B) drug control (normal rats treated with tangeretin alone), (C) diabetic control, (D) diabetic rats treated with tangeretin, (E) diabetic rats treated with metformin.
administration of tangeretin and glibenclamide to diabetic rats enhanced glucose utilization by increasing the activity of glucose6-phosphate dehydrogenase as evidenced by increased glycogen content in the liver. Glycogen, a branched polymer of glucose residues synthesized by the enzyme glycogen synthase, is the primary intracellular storable form of glucose and its quantity in various tissues is a direct manifestation of insulin activity as insulin supports intracellular glycogen deposition by stimulating glycogen synthase and inhibiting glycogen phosphorylase (Pushparaj et al. 2000). In the current study, the glycogen content was decreased in the liver muscle of diabetic rats. But, the glycogen content was found to be significantly increased in the liver of diabetic rats upon treatment with tangeretin and glibenclamide. This result indicates that tangeretin treatment is able to produce more insulin from pancreatic  cells and increase the glycogen content in the liver of diabetic rats by increasing the activity of glycogen synthase and inhibiting activity of glycogen phosphorylase. Conclusion According to the findings we have obtained in our study, the administration of tangeretin (100 mg/kg body weight) to diabetic rats restored the activities of key enzymes involved in the metabolism of glucose and glycogen. Therefore, this result reveals
that tangeretin enhances the glycolytic enzymes and controls the glucose metabolism in the hepatic tissues of diabetic rats by stimulating insulin production from existing beta cells of pancreas by its antioxidant potential. The effect produced by tangeretin on various parameters comparable to that of glibenclamide. Further studies are needed to explore the molecular mechanisms of tangeretin in altering hormone secretion. References Ainscow, E.K., Zhao, C., Rutter, G.A., 2000. Acute overexpression of lactate dehydrogenase – a perturbs beta-cell mitochondrial metabolism and insulin secretion. Diabetes 49, 1149–1155. Alberti, K.G.M.M., Press, C.M., 1982. The biochemistry and the complications of diabetes. In: Keen, H., Jarrett, J. (Eds.), Complications of Diabetes, vol. 43. Arnold Publishers, London, pp. 231–270. Anand, P., Murali, K.Y., Tandon, V., Murthy, P.S., Chandra, R., 2010. Insulinotropic effect of cinnamaldehyde on transcriptional regulation of pyruvate kinase, phosphoenol pyruvate carboxykinase and GLUT4 translocation in experimental diabetic rats. Chemico Biological Interactions 186, 72–81. Bisse, E., Abragam, E.C., 1985. New less temperature sensitive, microchromato graphic method for the separation and quantitation of glycosylated haemoglobin using a non cyanide buffer system. Journal of Chromatography 34, 81–91. Brandstrup, N., Kirk, J.E., Bruni, C., 1957. Determination of hexokinase in tissues. Journal of Gerontology 12, 166–171. Cheng, Z., Surichan, S., Ruparelia, K., Arroo, R., Boarder, M.R., 2011. Tangeretin and its metabolite 4 -hydroxytetramethoxyflavone attenuate EGF-stimulated cell cycle progression in hepatocytes; role of inhibition at the level of mTOR/p70S6K. British Journal of Pharmacology 162, 1781–1791.
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Pari, L., Rajarajeswari, N., 2009. Efficacy of coumarin on hepatic key enzymes of glucose metabolism in chemical induced type 2 diabetic rats. Chemico Biological Interaction 181, 292–296. Pari, L., Saravanan, R., 2005. Succinic acid monoethyl ester and metformin regulates carbohydrate metabolic enzymes and improves glycemic control in streptozotocin-nicotinamide induced type 2 diabetic rats, 4., pp. 132–137. Paulson, D.J., 1997. The diabetic heart is more sensitive to ischemic injury. Cardiovascular Research 34, 104–112. Pederson, B.A., Schroeder, J.M., Parker, G.E., Smith, M.W., DePaoli-Roach, A.A., Roach, P.J., 2005. Glucose metabolism in mice lacking muscle glycogen synthase. Diabetes 54, 3466–3473. Pogson, C.I., Denton, R.M., 1967. Effect of alloxan diabetes, starvation and refeeding on glycolytic kinase activities in rat epididymal adipose tissue. Nature 216, 156–157. Pushparaj, P.N., Tan, C.H., Tan, B.K.H., 2000. Effects of Averrhoabilimli leaf extract on blood glucose and lipids in streptozotocin diabetic rats. Journal of Ethnopharmacology 72, 69–76. Roden, M., Bernroide, E., 2003. Hepatic glucose metabolism in humans – its role in health and disease. Best Practice & Research: Clinical Endocrinology & Metabolism 17, 365–383. Sabu, M.C., Smitha, K., Kuttan, R., 2002. Anti-diabetic activity of green tea polyphenols and their role in reducing oxidative stress in experimental diabetes. Journal of Ethnopharmacology 83, 109–116. Shanmugam, K.R., Mallikarjuna, K., Nishanth, K., Kuo, C.H., Sathyavelu Reddy, K., 2011. Protective effect of dietary ginger on antioxidant enzymes and oxidative damage in experimental diabetic rat tissues. Food Chemistry 124, 1436–1442. Shulman, G.I., 2000. Cellular mechanisms of insulin resistance. Journal of Clinical Investigations 106, 171–176. Srinivasan, K., Ramarao, P., 2007. Animal model in type 2 diabetes: an overview. Indian Journal of Medical Research 125, 451–472. SubashBabu, P., Prabuseenivasan, P., Ignacimuthu, S., 2007. Cinnamaldehyde a potential antidiabetic agent. Phytomedicine 14, 15–22. Takano, K., Tabata, Y., Kitao, Y., et al., 2007. Methoxyflavones protect cells against endoplasmic reticulum stress and neurotoxin. American Journal of Physiology – Cell Physiology 292, C353–C361. Taylor, R., Agius, L., 1988. The biochemistry of diabetes. Biochemical Journal 250, 625–640. Trinder, P., 1969. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Annals of Clinical Biochemistry 6, 24–27. Wajngot, A., Chandramouli, V., Schumann, W.C., Ekberg, K., Jones, P.K., Efendic, S., Landau, B.R., 2001. Quantitative contributions of gluconeogenesis to glucose production during fasting in type 2 diabetes mellitus. Metabolism 50, 47–52. World Health Organization, 2009. Prevalence Data of Diabetes Worldwide. Xu, G.H., Chen, J.C., Liu, D.H., et al., 2008. Minerals, phenolic compounds and antioxidant capacity of citrus peel extract by hot water. Journal of Food Science 73, C11–C18. Yamada, K., Noguchi, T., 1999. Nutrient and hormonal regulation of pyruvate kinase gene expression. Biochemistry Journal 337, 1–11. Yoon, J.H., Lim, T.G., Lee, K.M., et al., 2011. Tangeretin reduces ultraviolet B (UVB)-induced cyclooxygenase-2 expression in mouse epidermal cells by blocking mitogen-activated protein kinase (MAPK) activation and reactive oxygen species (ROS) generation. Journal of Agricultural and Food Chemistry 59, 222–228.
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Effect of tangeretin, a polymethoxylated flavone on glucose metabolism in streptozotocin-induced diabetic rats Ramalingam Sundaram a , Palanivelu Shanthi b , Panchanatham Sachdanandam a,∗ a Department of Medical Biochemistry, Dr. ALM P-G, Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, Tamil Nadu 600113, India b Department of Pathology, Dr. ALM P-G, Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, Tamil Nadu 600113, India
a r t i c l e
i n f o
Article history: Received 29 August 2013 Received in revised form 11 November 2013 Accepted 26 January 2014 Keywords: Tangeretin Diabetes Carbohydrate metabolism Cardiovascular disease
a b s t r a c t The present study was designed to evaluate the antihyperglycemic potential of tangeretin on the activities of key enzymes of carbohydrate and glycogen metabolism in control and streptozotocin induced diabetic rats. The daily oral administration of tangeretin (100 mg/kg body weight) to diabetic rats for 30 days resulted in a significant reduction in the levels of plasma glucose, glycosylated hemoglobin (HbA1c) and increase in the levels of insulin and hemoglobin. The altered activities of the key enzymes of carbohydrate metabolism such as hexokinase, pyruvate kinase, lactate dehydrogenase, glucose-6phosphatase, fructose-1,6-bisphosphatase, glucose-6-phosphate dehydrogenase, glycogen synthase and glycogen phosphorylase in liver of diabetic rats were significantly reverted to near normal levels by the administration of tangeretin. Further, tangeretin administration to diabetic rats improved hepatic glycogen content suggesting the antihyperglycemic potential of tangeretin in diabetic rats. The effect produced by tangeretin on various parameters was comparable to that of glibenclamide – a standard oral hypoglycemic drug. Thus, these results show that tangeretin modulates the activities of hepatic enzymes via enhanced secretion of insulin and decreases the blood glucose in streptozotocin induced diabetic rats by its antioxidant potential. © 2014 Elsevier GmbH. All rights reserved.
Introduction Diabetes mellitus (DM) is a chronic metabolic disease with the highest rates of prevalence and mortality worldwide that is caused by an absolute or relative lack of insulin and or reduced insulin activity, which results in hyperglycemy and abnormalities in carbohydrate, protein, and fat metabolism (Kamtchouing et al. 2006; Fatima et al. 2010). The World Health Organization (WHO) estimates that more than 220 million people worldwide have diabetes, and this number is liable to double by 2030 (WHO 2009). The chronic hyperglycaemia of diabetes is associated with damage, dysfunction and failure of various organs such as kidneys, retina, heart, liver, peripheral and central nervous system (Shanmugam et al. 2011). Recently, much attention has been focused on screening of products from natural sources, such as flavonoids, that may be beneficial for reducing the risk for metabolic syndrome (Sabu et al. 2002; Jung
∗ Corresponding author. Tel.: +91 9884855505. E-mail addresses: [email protected] (P. Shanthi), [email protected] (P. Sachdanandam). 0944-7113/$ – see front matter © 2014 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2014.01.007
et al. 2004), because products from plant sources are usually considered to be less toxic and have fewer side effects than products from synthetic sources. Tangeretin is a polymethoxylated flavones abundant in the citrus fruit rinds, including mandarin orange, Poncirus trifoliate Raf. Tangeretin plays an important role in preventing various diseases including cancer, oxidative stress, and inflammation (Yoon et al. 2011; Xu et al. 2008). In addition to its anti-oxidant effects, tangeretin has been reported to inhibit the growth of hepatocytes both in vitro and in vivo via inhibition of mTOR/p70S6 kinase (Cheng et al. 2011). Regarding tangeretin mediated effects on neuronal disease, a number of studies have shown that tangeretin reduces dopaminergic neurotoxin-induced neuronal injury and prevents tunicamycin-induced cell death in mice through an increase in glucose-regulated protein (GRP)78 and heme oxygenase (HO)-1 expression in renal tubular epithelium (Takano et al., 2007). A recent study investigating the anti-inflammatory effects of citrus fruit peels showed that tangeretin had a beneficial effect on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cells, which may provide protection against disease resulting from excessive NO production (Choi et al. 2007).
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OCH 3 O
Experimental design
H3CO H3CO
O OCH 3
OCH 3
Fig. 1. Tangeretin.
Considering the wide variety of pharmacological action of tangeretin, the present study was undertaken to explore the tangeretin on key hepatic enzyme in streptozotocin induced diabetic rats. The effect of tangeretin was compared with conventional antidiabetic agent glibenclamide. The chemical structure of tangeretin was given in Fig. 1.
Materials and methods Sources of chemicals All fine chemicals including streptozotocin were purchased from Sigma Chemical Company (St. Louis, MO, USA). All other chemicals used were of good quality and analytical grade and obtained from Himedia, Mumbai, India.
Animals Male albino Wistar rats weighing 180–200 g m body weight were procured from the Central Animal House Facility, University of Madras, Taramani Campus, Chennai, Tamil Nadu, India. They were maintained at an ambient temperature of 25 ± 2 ◦ C and 12/12 h of light/dark cycle. Animals were given standard commercial rat chow and water ad libitum and housed under standard environmental conditions throughout the study. The experiments were conducted according to the ethical norms approved by the Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines.
Experimental induction of diabetes Diabetes was induced in overnight fasted experimental rats by single intraperitoneal injection of streptozotocin (60 mg/kg bodyweight) dissolved in freshly prepared 0.2 ml of 0.1 mol/l citrate buffer, pH 4.5. Streptozotocin injected animals were allowed to drink 20% glucose solution overnight to overcome the initial druginduced hypoglycemic mortality. Control rats were injected with the vehicle (0.2 ml of 0.1 mol/l citrate buffer, pH 4.5) alone. After 96 h, plasma glucose was determined and those rats with fasting blood glucose greater than 300 mg/dl were used in the present study. In diabetic rats, the treatment was started six weeks after the onset of diabetes because, in many studies, the duration of diabetes required to induce cardiac dysfunction in experimental rats has been found to be six weeks (Paulson 1997). Oral glucose tolerance test (OGTT) was performed according to the method of Du Vigneaud and Karr (1925). After overnight fasting, ‘0 minute blood sample (0.2 ml) was taken from control and experimental rats. Without delay, a glucose solution (2 g/kg body weight) was administered by oral gavage. Blood samples were taken at 30, 60, 90 and 120 min after glucose administration. Blood samples were collected with potassium oxalate and sodium fluoride and glucose levels were determined by the kit method of Trinder (1969).
The animals were randomly divided into seven groups of six animals in each (30 diabetic surviving and 12 normal). Group I: Control animals (normal healthy control rats received intra gastrically 0.5 ml of 0.9% saline for 30 days. Group II: Drug control (normal healthy control rats received intra gastrically tangeretin (100 mg/kg b.w.) dissolved in 0.5 ml of 0.9% saline for 30 days. Group III: Diabetic control rats. Group IV: Diabetic rats received intra gastrically tangeretin (25 mg/kg b.w) dissolved in 0.5 ml of 0.9% saline for 30 days. Group V: Diabetic rats received intra gastrically tangeretin (50 mg/kg b.w) dissolved in 0.5 ml of 0.9% saline for 30 days. Group VI: Diabetic rats received intra gastrically tangeretin (100 mg/kg b.w) dissolved in 0.5 ml of 0.9% saline for 30 days. Group VII: Diabetic rats received intra gastrically glibenclamide (5 mg/kg b.w) dissolved in 0.5 ml of 0.9% saline for 30 days. At the end of the treatment period (75 days), the rats were fasted overnight, anaesthetized and sacrificed by cervical decapitation. The blood was collected with or without EDTA for plasma or serum separation, respectively. The liver tissue was dissected out, washed in ice-cold saline, and weighed. Tissue was minced and homogenized (10%, w/v) with 0.1 M Tris–HCl buffer (pH 7.4) and centrifuged (3000 × g for 10 min). The resulting supernatant was used for enzyme assays. Body weights of all the animals were recorded prior to the treatment and sacrifice. Food and water intake of all groups of animals were monitored on a daily basis for 30 days at a fixed time. Fixed amount of rat chow and fluid was given to each rat and replenished the next day. Biochemical analysis The level of plasma glucose was estimated spectrophotometrically using commercial diagnostic kit (Agappe Diagnostics Pvt. Ltd., Kerala, India) Trinder (1969). Plasma insulin level was assayed by enzyme linked immunosorbent assay kit (ELISA) (Boehringer Mannheim kit). Hemoglobin and HbA1C was estimated by diagnostic kit (Agappe Diagnostic Pvt. Ltd., India) Bisse and Abragam, (1985). The estimation of protein was carried out by the method of Lowry et al. (1951). Hepatic hexokinase activity was assayed by the method of Brandstrup et al. (1957). Glucose-6-phosphate dehydrogenase was assayed by the method of Ellis and Kirkman (1961). Glucose-6-phosphatase was assayed by the method of Koide and Oda (1959). Fructose-1,6-bisphosphatase activity was measured by the method of Gancedo and Gancedo (1971), pyruvate kinase activity was estimated by the method of Pogson and Denton (1967), lactate dehydrogenase activity was estimated by the method of King (1965), glycogen synthase activity was estimated by the method of Leloir and Goldemberg (1962), glycogen phosphorylase activity was estimated by the method of Cornblath et al. (1963). Another portion of wet liver glycogen content was estimated by the method of Morales et al. (1973). Histopathological studies Pancreatic tissues were harvested from the sacrificed animals and were fixed in 10% neutral buffered formalin solution, dehydrated in ethanol and embedded in paraffin. Sections of 5 m thickness were prepared using a rotary microtome and stained with hematoxylin and eosin dye and mounted in neutral deparaffinated xylene medium for microscopic observations.
R. Sundaram et al. / Phytomedicine 21 (2014) 793–799 Table 1 Dose-dependent effects of tangeretin on changes in plasma glucose and insulin in normal and diabetic rats. Group
Glucose (mg/dl)
Control Normal + tangeretin (100 mg/kg b.w.) Diabetic control Diabetic + tangeretin (25 mg/kg b.w.) Diabetic + tangeretin (50 mg/kg b.w) Diabetic + tangeretin (100 mg/kg b.w.) Diabetic + glibenclamide (5 mg/kg)
90.33 87.0 323.33 280.93 220.37 133.16 128.33
± ± ± ± ± ± ±
8.64 6.78 24.42b 10.47c 14.16d 10.04e 7.20f
Insulin (U/ml) 17.73 18.76 6.26 8.56 11.25 15.98 16.35
± ± ± ± ± ± ±
1.24 1.52 0.53b 0.51c 0.73d 1.49e 1.23f
Values are given as mean ± S.D. for six animals in each group (n = 6), Values are considered significantly different at p < 0.05 with post hoc LSD test. a Control vs drug control (tangeretin alone treated rat). b Control rat vs diabetic rat. c Diabetic rat vs tangeretin 25 mg/kg. d Diabetic rat vs tangeretin 50 mg/kg. e Diabetic rat vs tangeretin 100 mg/kg. f Tangeretin (100 mg/kg) treated diabetic rat vs glibenclamide treated diabetic rats (5 mg/kg).
Statistical analysis The results are expressed as mean ± standard deviation (S.D.). Differences between groups were assessed by ANOVA using the SPSS software package for Windows. Post hoc testing was performed for inter-group comparisons using the least significance difference (L.S.D.) p-values < 0.05 were considered as significantly altered. Results Estimation of plasma glucose, insulin and glucose tolerance Table 1 shows the levels of plasma glucose, insulin in normal and experimental rats. A significant increase in plasma glucose and a decrease in insulin levels were observed in diabetic rats. However, administration of tangeretin to diabetic rats significantly decreased the blood glucose levels to near normal. There was no significant change in normal rats treated with tangeretin. Results of OGTT conducted on control and different experimental groups are shown in Fig. 2. After the oral dose of glucose in normal control rats the blood glucose reached the fasting levels at 2 h. In diabetic control rats, the blood glucose levels remained higher even after 2 h. Treatment with tangeretin showed a significant decrease in blood glucose levels. The maximum glucose lowering effect of tangeretin was observed at a dose of 100 mg/kg body weight than the other two doses. Therefore, further studies were carried out with this dose. Determination of hemoglobin and HbA1C The levels of hemoglobin (Hb) and glycosyated hemoglobin (HbA1C) in control and experimental animals were depicted in Table 2. Decreased Hb and an increase in HbA1C were observed in diabetic rats. These values improved toward near normal on treatment with tangeretin and glibenclamide. However, no significant variations were found in the control rats treated with tangeretin alone for a period of 30 days. Changes in body weight, organ weight, food and water intake The changes in the body weight, liver weight, food and water intake of control and experimental animals were represented in Table 3. The baseline body weight at the beginning of the study was similar in all groups. At the end of the experimental period, the body weight and liver weight was significantly decreased
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in diabetic rats when compared to control rats. However, oral administration of tangeretin and glibenclamide to diabetic rats reversed the body weight and liver weight changes to near normal. Normal rats treated with tangeretin showed no significant changes in the body weight and liver weight. Diabetic animals also showed signs of polyuria, polydipsia and polyphagia. The food and water intake increased in diabetic rats compared with control rats (Table 3). Administration of tangeretin and glibenclamide significantly decreased the food and water intake in diabetic rats. Activities of carbohydrate metabolic enzymes Table 4 portrays the changes in the activities of carbohydrate metabolizing enzymes in the liver of control and experimental rats. The activities of hexokinase, pyruvate kinase and glucose-6phosphate dehydrogenase were significantly decreased whereas the activities of lactate dehydrogenase. Glucose-6-phosphatase and fructose-1,6-biphosphatase were significantly increased in diabeticrats when compared to normal control rats. However, upon treatment with tangeretin and glibenclamide to diabetic rats reversed the activities of these hepatic key enzymes to near normal. Glycogen content and activities of glycogen metabolic enzymes Liver glycogen metabolism of control and experimental animals were shown in Table 5. A significant decline in the glycogen level as well as in the glycogen synthase activity and a concomitant increase in the activity of glycogen phosphorylase were noted in the liver of diabetic rats. Oral treatment with tangeretin and glibenclamide to diabetic groups of rats reinstated the level of glycogen and the activities of glycogen synthase and glycogen phosphorylase to near normal when compared to control rats. Histological examination of pancreas Fig. 3 represents the microphotographs of H&E staining of pancreatic tissues of normal control and experimental rats. Histopathological examination showed that streptozotocin and high fat diet administration elicited severe injury in pancreatic ß-cells, such as a decrease of islet cells’ number and vascular degenerative changes in the islets (Fig. 3C). The pancreas of the control and drug control rat showed normal islets and well organized dark beta cells (Fig. 3A and B). Both the tangeretin and glibenclamide treated diabetic rats showed the regenerating islets an increase in the islets as compared with the diabetic control group (Fig. 3D and E). Discussion Streptozotocin (STZ)-induced hyperglycemia in animals is considered to be a good model for the preliminary screening of agents active against diabetes and is widely used (Ivorra et al. 1989). In experiment with many animal species, streptozotocin produces permanent diabetes that impersonates the pathological status of human diabetes (Larson et al., 2002). Therefore, streptozotocin induced diabetes is reproducible, convenient and can produce diabetes of graded severity suitable for experimental diabetes (Srinivasan and Ramarao 2007). The mechanism by which streptozotocin brings about its diabetic state includes selective destruction of pancreatic beta cells which make cells less active and leading to poor sensitivity of insulin for glucose uptake by tissues which causes hyperglycemia (Jacot and Assal 1989; Marles and Farnsworth 1995). The persistent supra-physiological level of glucose non-enzymatically reacts with hemoglobin to form increased glycosylated hemoglobin which is a standard biochemical marker for the diagnosis of ambient glycemia during a period
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Fig. 2. Oral glucose tolerance test.
Table 2 Effect of tangeretin on the levels of Hb and HbA1c in normal and diabetic rats. Parameters
Normal control
Normal + tangeretin (100 mg/kg b.w.)
Diabetic control
Diabetic + tangeretin (100 mg/kg b.w.)
Diabetic + glibenclamide (5 mg/kg bw)
Hb (g/dl) HbA1c (mg/g of Hb)
14.38 ± 1.04 4.75 ± 0.33
13.96 ± 1.07 4.22 ± 0.31
7.44 ± 0.54b 13.41 ± 1.05b
12.01 ± 0.93c 6.18 ± 0.58c
12.66 ± 1.21 5.83 ± 0.41
Values are given as mean ± S.D. for six animals in each group (n = 6), Values are considered significantly different at p < 0.05 with post hoc LSD test. a Control vs Drug control (Tangeretin alone treated rats). b Control rats vs Diabetic rats. c Diabetic rats vs Tangeretin treated diabetic rats. d Tangeretin treated diabetic rats vs Glibenclamide.
of 3-month (Alberti et al. 1982). The oral administration of tangeretin and glibenclamide to diabetic rats for 30 days significantly reversed plasma insulin, blood glucose, glycosylated hemoglobin and hemoglobin. Based on the results obtained from this study, it can be concluded that the elevated levels of pancreatic insulin may have enhanced the glucose utilization by peripheral tissues of diabetic rats either by promoting glucose uptake and metabolism, or by inhibiting hepatic gluconeogenesis and decreased blood glucose levels. In previous investigations the diabetic animals displayed the following characteristics polyuria, increased water intake, dehydration, weight loss and muscle wasting, excessive hair loss
and scaling, diarrhea, cataracts and increased food intake (Pari and Rajarajeswari 2009). Decrease in body weight of diabetic rats is due to catabolism of fats and proteins. Due to insulin deficiency the protein content is decreased in muscular tissue by proteolysis (SubashBabu et al. 2007). On oral administration of tangeretin and glibenclamide prevented the loss of body weight, excess of food and fluid intake in diabetic rats by controlling the hyperglycemia. Liver is a general metabolic organ that plays a pivotal role in glycolysis and gluconeogenesis. In the present study, a marked decrease in liver weight was observed in diabetic rats. This could be due to an increased breakdown of glycogen, protein degradation and increased gluconeogenesis. This result corroborates with
Table 3 Body weight, food and fluid intake in normal and diabetic rats before and after oral treatment with tangeretin. Parameters
Normal control
Body weight initial (g) Body weight final (g) Liver weight (g) Food intake after (g/rat/day) Fluid intake before (ml/rat/day) Fluid intake after (ml/rat/day)
244.6 467.1 12.07 14.58 72.16 70.66
± ± ± ± ± ±
10.57 12.08 1.15 1.49 5.49 5.35
Normal + tangeretin (100 mg/kg b.w.) 240.8 464 12.42 14.13 71.5 68.83
± ± ± ± ± ±
9.70 13.14 0.92 0.82 6.22 4.87
Diabetic control 240.6 224.6 7.55 65.03 128.1 137.5
± ± ± ± ± ±
8.75 8.28b 0.70b 5.69b 6.79 8.24b
Diabetic + tangeretin (100 mg/kg b.w.) 242.6 442.5 10.71 27.36 114.3 88.66
± ± ± ± ± ±
6.18 16.35c 0.76c 1.88c 8.64 5.20c
Diabetic + glibenclamide (5 mg/kg bw) 246.3 447.3 11.01 26.10 112 85
Values are given as mean ± S.D. for six animals in each group (n = 6). Values are considered significantly different at p < 0.05 with post hoc LSD test. a Control vs Drug control (Tangeretin alone treated rats). b Control rats vs Diabetic rats. c Diabetic rats vs Tangeretin treated diabetic rats. d Tangeretin treated diabetic rats vs Glibenclamide.
± ± ± ± ± ±
8.77 15.25 0.73 1.64 7.66 6.03
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Table 4 Effect of tangeretin on the activities of carbohydrate metabolizing enzymes in the liver of normal and diabetic rats. Parameters
Normal control
Normal + tangeretin (100 mg/kg b.w.)
Diabetic control
Diabetic + tangeretin (100 mg/kg b.w.)
Diabetic + glibenclamide (5 mg/kg bw)
Hexokinase Lactate dehydrogenase Pyruvate kinase Glucose 6 phosphatase Fructose 1,6 bisphopatase Glucose 6 phophatase
254.6 ± 14.5 239 ± 13.28 8.97 ± 0.80 1040 ± 15.79 494.3 ± 21.62 548.5 ± 27.48
257.1 ± 10.70 235.5 ± 13.32 9.44 ± 0.76 1037 ± 16.14 492.1 ± 23.43 551.6 ± 27.78
126.5 ± 11.2b 438.3 ± 18.98b 3.23 ± 0.31b 1925 ± 31.10b 805.6 ± 30.91b 266.5 ± 15.93b
205.5 ± 12.9c 268.8 ± 10.20c 7.42 ± 0.70c 1141 ± 28.88c 518.8 ± 25.95c 496.1 ± 11.73c
208.8 ± 8.56 265.6 ± 9.33 7.99 ± 0.76 1138 ± 27.78 515.1 ± 26.07 502.1 ± 11.12
Hexokinase – moles glucose-6-phosphatase formed in hour/mg of protein, lactate dehydrogenase – moles of pyruvate formed/hour/mg of protein, pyruvate kinase – moles of pyruvate formed in minute/mg of protein, glucose 6 phosphatase, fructose 1,6 bisphopatase – moles phosphate liberated in h/mg of protein, glucose-6-phophate dehydrogenase – Units/min per mg of protein. Values are given as mean ± S.D. for six animals in each group (n = 6). Values are considered significantly different at p < 0.05 with post hoc LSD test. a Control vs Drug control (Tangeretin alone treated rats). b Control rats vs Diabetic rats. c Diabetic rats vs Tangeretin treated diabetic rats. d Tangeretin treated diabetic rats vs Glibenclamide.
previous report (Anand et al. 2010). Tangeretin and glibenclamide, treatment has elevated the liver weight in diabetic rats by reversing gluconeogenesis. Further, it was confirmed by elevated levels of glycogen synthase and declined the levels of glycogen phospharylase in the liver of diabetic treated rats which in turn increase the glycogen content in liver. Elevated endogenous glucose production is a common abnormality associated with diabetes that, in concurrence with deprived pancreatic function and reduced glucose clearance, contributes to the hyperglycemia characteristic of the disease, diabetes (Wajngot et al. 2001). Insulin regulates the metabolism by modulating the uptake and utilization of glucose in target organs such as liver, kidney, skeletal muscle and adipose tissue by controlling the activities of numerous metabolic enzymes. Hexokinase is insulin dependent and one of the key enzymes of carbohydrate metabolism which involve the phosphorylation of glucose into glucose-6-phosphate and its level was significantly decreased in the liver of diabetic rats, this may be the reason for the diminished consumption of glucose in the system and increased blood sugar levels due to lack of insulin and loss of insulin receptor (Roden and Bernroide 2003). Administration of tangeretin and glibenclamide to diabetic rats significantly reduces the levels of plasma glucose which activates hexokinase and increase glycolysis and utilization of glucose for energy production via enhanced secretion of insulin from remnant pancreatic  cells. Liver-pyruvate kinase activity decreases as the result of diabetes and increases by the administration of insulin to diabetic rats (Yamada and Noguchi 1999). The altered activity during diabetic conditions could be expected to diminish the metabolism of glucose and ATP production. Hence, the observed decline in the activity of PK in the liver of diabetic rats promptly responsible for the reduced glycolysis and amplified gluconeogenesis signifying that these two pathways are altered in diabetes (Taylor and Agius 1988; Kavanagh et al. 2004). The treatment of tangeretin and glibenclamide to diabetic rats showed a notable increase in plasma
insulin that induces a decrease in ATP, a known allosteric inhibitor of PK, thereby increases the PK activity to near normalcy. Lactate dehydrogenase (LDH) is a terminal glycolytic enzyme that plays an indispensable role in the interconversion of pyruvate to lactate to yield energy under anaerobic conditions and the reaction occurs in both cytosolic and mitochondrial compartments (Ainscow et al. 2000). This enzyme is a tetramer, which can be composed of two different kinds of subunits: M (muscle type) and H (heart type) and the biosynthesis of each of these subunits is apparently controlled by separate genes. LDH activity is found to be altered by insulin, glucose, NADH, as well as increases in mitochondrial membrane potential, cytosolic free ATP and cytosolic free Ca2+ (Shulman 2000). The decreased activity of LDH in tissues could be important to ensure that a high proportion of both pyruvate and NADH, supplied by glycolysis, is subsequently oxidized by mitochondria. Indeed, elevated LDH levels observed in the experimental diabetic animals’ are associated with impaired glucose-stimulated insulin secretion (Pari and Saravanan 2005). Thus, increased activity of LDH interferes with normal glucose metabolism and insulin secretion in the  cells of pancreas and it may therefore be directly responsible for insulin secretory defects in diabetes. However, treatment with tangeretin and glibenclamide to diabetic rats reverted the LDH activity to near normalcy most probably by regulating the proportion of pyruvate and NADH thereby promoting the mitochondrial oxidation of (pyruvate) glucose. Glucose-6-phosphatase and fructose-1,6-bisphosphatase are the important enzymes in regulating of gluconeogenic pathway. The activities of glucose-6-phosphatase and fructose-1,6bisphosphatase were increased in the liver of diabetic rats (Pederson et al. 2005). The activities of the two enzymes may be due to the increased synthesis of enzymes contributing to the increased glucose production during diabetes by the liver (Mitra et al. 1995). Tangeretin inhibits gluconeogensis by inhibiting the activity of glucose-6-phosphatase and fructose-1,6-bisphosphatase. Oral
Table 5 Effect of tangeretin on the levels of glycogen content and activities of glycogen synthase and glycogen phosphorylase in liver tissues of normal and diabetic rats. Parameters
Normal control
Normal + tangeretin (100 mg/kg b.w.)
Diabetic control
Diabetic + tangeretin (100 mg/kg b.w.)
Diabetic + glibenclamide (5 mg/kg bw)
Liver glycogen (mg/g tissue) Glycogen synthase ( moles of UDP formed/h/mg protein) Glycogen phosphorylase ( moles Pi liberated/h/mg protein)
56.34 ± 5.11 860.5 ± 11.7
57.02 ± 5.30 862.5 ± 11.92
15.77 ± 1.45b 547.1 ± 18.25b
48.05 ± 4.14c 802.1 ± 16.24c
48.65 ± 4.30 805.8 ± 15.8
646.5 ± 12.7
648.8 ± 12.3
886.6 ± 23.59b
731.5 ± 17.32c
728.3 ± 16.6
Values are given as mean ± S.D. for six animals in each group (n = 6). Values are considered significantly different at p < 0.05 with post hoc LSD test. Control vs Drug control (Tangeretin alone treated rats). b Control rats vs Diabetic rats. c Diabetic rats vs Tangeretin treated diabetic rats. d Tangeretin treated diabetic rats vs Glibenclamide. a
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Fig. 3. Histopathological changes in pancreas of control and diabetic rats. All the sections are in H&E 40X. (A) Normal control, (B) drug control (normal rats treated with tangeretin alone), (C) diabetic control, (D) diabetic rats treated with tangeretin, (E) diabetic rats treated with metformin.
administration of tangeretin and glibenclamide to diabetic rats enhanced glucose utilization by increasing the activity of glucose6-phosphate dehydrogenase as evidenced by increased glycogen content in the liver. Glycogen, a branched polymer of glucose residues synthesized by the enzyme glycogen synthase, is the primary intracellular storable form of glucose and its quantity in various tissues is a direct manifestation of insulin activity as insulin supports intracellular glycogen deposition by stimulating glycogen synthase and inhibiting glycogen phosphorylase (Pushparaj et al. 2000). In the current study, the glycogen content was decreased in the liver muscle of diabetic rats. But, the glycogen content was found to be significantly increased in the liver of diabetic rats upon treatment with tangeretin and glibenclamide. This result indicates that tangeretin treatment is able to produce more insulin from pancreatic  cells and increase the glycogen content in the liver of diabetic rats by increasing the activity of glycogen synthase and inhibiting activity of glycogen phosphorylase. Conclusion According to the findings we have obtained in our study, the administration of tangeretin (100 mg/kg body weight) to diabetic rats restored the activities of key enzymes involved in the metabolism of glucose and glycogen. Therefore, this result reveals
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