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Effect of T. foenumgraecum on glycogen content of tissues and the key enzymes of carbohydrate metabolism
Journal of Ethnopharmacology 85 (2003) 237–242
Effect of T. foenumgraecum on glycogen content of tissues and the key enzymes of carbohydrate metabolism V. Vats, S.P. Yadav, J.K. Grover∗ Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110049, India Received 31 May 2002; received in revised form 20 December 2002; accepted 20 December 2002
Abstract The Indian traditional system of medicine prescribed plant therapies for diseases including diabetes mellitus called madhumeh in Sanskrit. One such plant mentioned in Ayurveda is Trigonella foenumgraecum (FG). In the present study, FG (1 g/kg PO) was assessed for its effect on glycogen levels of insulin dependent (skeletal muscle and liver), insulin independent tissues (kidneys and brain) and enzymes such as glucokinase (GK), hexokinase (HK), and phosphofructokinase (PFK). Administration of FG led to decrease in blood glucose levels by 14.4 and 46.64% on 15th and 30th day of the experiment. Liver and 2-kidney weight expressed as percentage of body weight was significantly increased in diabetics (P < 0.0005) versus normal controls and this alteration in the renal weight (P < 0.0005) but not liver weight was normalized by feeding of FG. Renal glycogen content increased by over 10 folds while hepatic and skeletal muscle glycogen content decreased by 75 and 68% in diabetic controls versus controls and these alteration in glycogen content was partly prevented by FG. Activity of HK, GK and PFK in diabetic controls was 35, 50 and 60% of the controls and FG partially corrected this alteration in PFK, HK and GK. © 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: T. foenumgraecum; Methi; Streptozotocin; Experimental diabetes; Carbohydrate metabolism
1. Introduction It is projected that incidence of diabetes is on rise. Present number of diabetics worldwide is 150 million and this is likely to increase to 300 million or more by the year 2025 (King et al., 1998). Reasons for this rise include increase in sedentary lifestyle, consumption of energy rich diet, obesity, higher life span, etc. (Yajnik, 2001). Though biguanides and sulfonylureas are valuable in treatment of diabetes mellitus, their use is restricted by their limited action, pharmacokinetic properties, secondary failure rates and accompanying side effects (Bailey et al., 1989). Moreover, these therapies only partially compensate for metabolic derangements seen in diabetics and do not necessarily correct the fundamental biochemical lesion (Taylor and Agius, 1988). Nature has been a source of medicinal treatments for thousands of years, and plants-based systems continue to play an essential role in the primary health care of 80% of the world’s underdeveloped and developing countries (King et al., 1998). ∗ Corresponding author. Tel.: +91-11-659-4897 (O)/461-5315 (R); fax: +91-11-686-2663. E-mail address: [email protected] (J.K. Grover).
Biguanides developed from a prototypic plant molecule is an excellent example of anti-diabetic drug development from plants. Thus, it is prudent in the current context to look for new and if possible more efficacious hits from the vast reserves of phytotherapy. Trigonella foenumgraecum Linn (FG) has been shown to possess hypoglycemic activity in experimental animals (Ghafghazi et al., 1977; Ribes et al., 1984, 1986; Swanston-Flatt et al., 1989; Ali et al., 1995; Khosla et al., 1995). However, very little is known about cellular and biochemical mechanism of the hypoglycemic or anti-hyperglycemic effect of FG. Although Raju et al. (2001) have shown that FG favorably effects glycolytic and gluconeogenic enzymes, they have worked with a whole seed powder diet. Since seeds have a very high fiber content, it is not possible to ascertain whether the anti-diabetic effect seen in their study was due to the effect of solid seed contents or some active pharmacological constituents. Therefore, this study was undertaken to assess the effect of FG on key enzymes of carbohydrate metabolism and glycogen content of various tissues. The dose was based on a previous pilot work that was undertaken to establish and assess the dose–response relationship (Vats et al., 2002).
0378-8741/03/$ – see front matter © 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00022-9
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2. Materials and methods
2.5. Biochemical and enzymatic estimations
2.1. Preliminary phytochemical evaluation
2.5.1. Plasma glucose Glucose levels were estimated by commercially available glucose kits based on glucose oxidase method (Trinder, 1969) (Autopak® , Bayer Diagnostics, Baroda).
It yielded moisture content of 10.3%, dry matter 89.7% (fiber 56.2%, protein 18.2%, mineral constituent 2.1%, alkaloidal content 0.011%, fixed oil in traces). The dose was 2 g/kg body weight. 2.2. Animals Male albino rats (150–200 g) were obtained from the experimental animal facility of the All India Institute of Medical Sciences. Before initiation and during the experiment, rats were fed standard chow diet. After randomization into various groups, the rats were acclimatized for 2–3 days in the new environment before initiation of experiment. Animals had free access to food and drinking water till before 30 min of sampling.
2.5.2. Hepatic phosphofructokinase activity (EC 2.7.1.11) The liver homogenate was prepared by grinding frozen tissue in ratio of 1:4 in 50 mM imidazole, 5 mM EDTA, 5 mM EGTA, 100 mM NaCl and 30 mM beta-mercaptoethanol (pH = 7). The homogenate was centrifuged at 12,000 × g for 15 min and supernatant was removed and stored in ice until assay. The phosphofructokinase activity was assayed spectrophotometrically by the method of Racker (1947). 2.5.3. Hepatic glucokinase activity The above-mentioned sample was used and enzyme activity was based on spectrophotometric method assaying glucose phosphorylation described by Pilkis (1975).
2.3. Experimental design All the animals were randomly divided into 3 groups with 10 animals in each group. Group I (CNT) were normal and used as controls. Group II (DCNT) were used as diabetic control. Group III was daily treated with FG after 10 days of standing hyperglycemia for 30 days. Streptozotocin (STZ) (Sigma, USA) was administered in Groups II–IV by a single intravenous injection of STZ (65 mg/kg) given intraperitoneally. STZ was first weighed in individual Eppendorf’s according to the weight of the animal, then solubilized with 0.2 ml saline (154 mM NaCl) just prior to injection. Rats exhibiting plasma glucose levels >300 mg/dl, 48 h after administration of STZ were included in the study. They were allowed a window period of 10 days before start of treatment. 2.4. Sample collection 2.4.1. Blood sample It was collected retro-orbitally from the inner canthus of the eye under light ether anesthesia using capillary tubes (Micro Hematocrit Capillaries, Mucaps). Blood was collected in fresh vials containing sodium fluoride and sodium oxalate as anti-coagulant/anti-glycolytic agents and plasma was separated in a T8 electric centrifuger (Remi Udyog, New Delhi) at 2000 rpm for 2 min. 2.4.2. Collection of organs After 30 days of daily feeding of FG orally (40 days after STZ), the animals were euthanized by overdose of intraperitoneal anesthesia and blood and tissue samples were collected for the assessment of plasma glucose, hepatic glucokinase (EC 2.7.1.1), hexokinase (EC 2.7.1.1), and phosphofructokinase (EC 2.7.1.11) content along with glycogen content in different tissues, i.e. liver, brain, heart and skeletal muscle.
2.5.4. Skeletal hexokinase activity The enzyme activity was determined by the method of Chou and Wilson (1975) and was based on the reduction of NADPH coupled with hexokinase measured spectrophotometrically at 340 nm. 2.5.5. Glycogen content The tissue sample was digested in hot concentrated 30% KOH, precipitated with ethanol, hydrolyzed and finally determined as glucose in the hydrolyzate as reducing sugar (Hassid and Abraham, 1957).
3. Results The basal levels of plasma glucose of the rats in all groups prior to STZ administration were not significantly different. However, 48 h after STZ administration, plasma glucose levels were significantly higher in the rats selected for the study. In contrast, non-diabetic controls remained persistently euglycemic throughout the course of the study. 3.1. Glucose levels The anti-hyperglycemic effect of FG on the blood sugar levels of diabetic rats is shown in Table 1. Controls rats did not show any significant variation in the blood glucose throughout the experimental period. Administration of STZ (65 mg/kg) led to over four-fold elevation of blood glucose levels (P < 0.001) which was maintained over a period of 5 weeks. After 4 weeks of daily treatment with FG led to a fall in blood sugar levels by 14.4 and 46.64% on 15th and 30th day of the experiment, respectively (P < 0.01 and P < 0.0001, respectively).
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Table 1 Effect of FG administration (30 days) on glucose levels (mg/%) in STZ (65 mg/kg) diabetic rats
CNT DCNT FG
0 day
10th day
25th day
40th day
78.76 ± 5.38 (n = 10) 77.90 ± 4.21 (n = 10) 80.87 ± 8.01 (n = 10)
81.29 ± 5.44 (n = 10) 325.43 ± 18.95∗ (n = 8) 322.86 ± 14.45 (n = 10)
82.11 ± 4.81 (n = 10) 318.28 ± 18.76∗ (n = 7) 276.21 ± 34.67∗ (n = 9) (14.44%)
84.39 ± 6.77 (n = 10) 321.53 ± 16.52∗ (n = 6) 172.27 ± 21.63∗∗ (n = 8) (46.64%)
CNT: control; DCNT: diabetic control. Values are given as mean ± S.D. for no. of animals indicated. Value in parenthesis indicates the percentage lowering of plasma sugar in comparison to the previous reading. Diabetic control was compared with normal control at the corresponding time interval and treated group was compared with the corresponding values at 48 h. ∗ Statistically significant at P < 0.01. ∗∗ Statistically significant at P < 0.0001.
Table 2 Effect of PM on body, liver and kidney weight in rats
Body weight on 0 day Body weight on 15th day Body weight on 30th day Liver weight on 30th day (g) Liver weight/100 g body weight on 30th day Kidneys weight on 30th day (g) Kidneys weight/100 g body weight on 30th day
CNT
DCNT
FG
163.1 ± 7.88 191.4 ± 9.14 213.2 ± 6.98 7.62 ± 0.68 3.53 ± 0.25 1.54 ± 0.08 0.71 ± 0.02
163.9 ± 10.98 165.7 ± 3.94∗∗∗ 168.7 ± 5.06∗∗∗ 7.45 ± 0.38 4.36 ± 0.16∗∗∗ 1.56 ± 0.04 0.92 ± 0.02∗∗∗
167.7 ± 13.6 175.4 ± 11∗ 189 ± 11∗∗,∗∗∗ 7.95 ± 0.59 4.18 ± 0.15 1.58 ± 0.09 0.84 ± 0.04∗∗
Abbreviations as for Table 1. Values are given as mean ± S.D. Value in parenthesis indicates the percentage increase or decrease vs. controls. Diabetic control was compared with normal control at the corresponding time interval and treated group was compared with diabetic controls. ∗ Statistically significant at P < 0.05. ∗∗ Statistically significant at P < 0.005. ∗∗∗ Statistically significant at P < 0.0005.
3.2. Body weight, renal and liver weight The effect of STZ and feeding FG on the body weight, renal and liver weight is shown in Table 2. There was no significant intra-group variation in the basal body weight on the 0 day of the experiment. While control rats gained significant weight in the 30-day period, there was no appreciable increase in the diabetic controls over the same period. On the other hand, treated rats gained significantly more weight than diabetic controls but the increase remained significantly lesser than the normal controls. There was no significant difference in the absolute weights of livers and kidneys among any of the experimental groups. However, liver and 2-kidney weight expressed as percentage of body weight was significantly increased in diabetics
(P < 0.0005) versus normal controls and this alteration in the renal weight but not liver weight was significantly reduced (P < 0.005) by feeding FG. 3.3. Glycogen content Glycogen content of various tissues (liver, skeletal muscle, heart, brain and kidneys) was estimated on the 30th day in normal controls, diabetic controls and FG-treated groups (see Table 3). As evident, the glycogen content decreased significantly by 75 and 68% in liver and skeletal muscle in diabetic controls as compared to non-diabetic controls. On the other hand renal glycogen content increased by over 10 folds in diabetic animals versus non-diabetic animals. Brain and heart glycogen content remained unaltered in diabetic
Table 3 Effect of FG administration (30 days) on glycogen content (mg/g tissue) of various tissues of STZ (65 mg/kg) diabetic rats Groups
Brain (mg/100 g)
Kidney (mg/g)
Heart (mg/g)
Muscle (mg/g)
Liver (mg/g)
CNT (n = 10) DCNT (n = 6) FG (n = 8)
40.34 ± 4.76 43.11 ± 6.41 40.33 ± 7.04
0.83 ± 0.22 11.5 ± 2.99∗∗ (↑1280%) 6.95 ± 1.48∗
1.81 ± 0.32 1.85 ± 0.58 1.8 ± 0.65
8.71 ± 1.22 2.81 ± 1.26∗∗ (↓68%) 4.6 ± 1.5∗
52.55 ± 5.22 13.25 ± 3.99∗∗ (↓75%) 30.15 ± 6.76∗∗
Abbreviations as for Table 1. Values are given as mean ± S.D. for no. of animals indicated. Value in parenthesis indicates the percentage increase or decrease vs. controls. Diabetic control was compared with normal control at the corresponding time interval and treated group was compared with diabetic control. ∗ Statistically significant at P < 0.005. ∗∗ Statistically significant at P < 0.0005.
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Table 4 Effect of administration (30 days) on levels of three important enzymes (%) involved in carbohydrate metabolism in STZ (65 mg/kg) diabetic rats Enzyme PFK GK HK
CNT (n = 10)
DCNT (n = 6)
FG (n = 8)
100 100 100
59.07∗∗∗
86.13∗∗ 73.20∗∗ 62.33∗∗
48.69∗∗ 35.56∗∗∗
Abbreviations as for Table 1. Values are given as mean ± S.D. for no. of animals indicated. Value in parenthesis indicates the percentage increase or decrease vs. controls. Diabetic control was compared with normal control at the corresponding time interval and treated group was compared with diabetic controls. ∗∗ Statistically significant at P < 0.001. ∗∗∗ Statistically significant at P < 0.0001.
animals while FG significantly attenuated the STZ-induced hepatic (P < 0.0005), and muscle (P < 0.005) renal (P < 0.005) glycogen content. 3.4. Hepatic enzymes The diabetic controls showed significant decrease in the values of PFK, GK and HK (see Table 4). The respective percentage decrease was approximately 40, 50 and 65% as compared to non-diabetic control values. As compared to diabetic controls, FG significantly altered these enzymes favorably but could not normalize them to the control values.
4. Discussion The present study was carried out to determine effect of feeding of FG on the key enzymes involved in carbohydrate metabolism in STZ (65 mg/kg) rats and glycogen content of insulin dependent (skeletal muscle and liver) and independent tissues (kidneys and brain). Diabetes mellitus is characterized by partial or total deficiency of insulin resulting in derangement of carbohydrate metabolism and a decrease in enzymatic activity of glucokinase, hexokinase and phosphofructokinase resulting in depletion of liver and muscle glycogen (Murphy and Anderson, 1974). GK and PFK are the most sensitive indicators of the glycolytic pathway in the diabetic state (Steiner and King, 1964). Since insulin administration normalizes these alterations in the enzymatic activities (Weber et al., 1966), these enzymes represent a method to assess peripheral utilization of glucose. In the present case, diabetic rats showed a significant reduction in all the three enzymes in comparison to the normal controls. Liver plays an important role in buffering the postprandial hyperglycemia and is involved in synthesis of glycogen. Diabetes mellitus is known to impair the normal capacity of the liver to synthesize glycogen (Hornbrook, 1970; Migliorini, 1971; Whitton and Hems, 1975). However, after food, diabetic animals fail to synthesize glycogen from glucose and gluconeogenic precursors. Synthase phosphatase activates glycogen synthase resulting in glycogenesis and
this activation appears to be defective in STZ-induced diabetic animals (Bishop, 1970; Tan and Nuttall, 1976; Golden et al., 1979). This inhibition of synthase phosphatase is almost complete after 3 days in alloxan-diabetic rats and in 1–2 weeks in STZ diabetes (Langdon and Curnow, 1983). Therefore, the drug treatment was started 10 days after STZ administration and continued for 4 weeks. STZ-induced diabetic animals tend to show renal hypertrophy. In the present case also, diabetic rats showed 29% increase in 2-kidney versus body weight ratio in comparison to controls. This degree of renal enlargement is lesser than 62 and 52% reported previously (Rasch, 1980; Nielsen et al., 1999). Probably degree of increase in renal weight is a time-dependent phenomenon as duration in the above-mentioned two studies was 6 and 2 months, respectively, as compared to 1 month in the present case. Forty days after STZ injection, GK and PFK levels of the diabetic rats were 48.69 and 59.07%, respectively of the controls and this is similar to previous finding (Steiner and King, 1964) in which both the dose of STZ and duration of diabetes is comparable with the present study. An increase in the activity of glucokinase and PFK in the FG-treated rats implies that cellular entry of glucose was facilitated by FG, which in turn stimulated the activity of these enzymes. This glucose influx could either be due to an insulin releasing or direct insulinomimetic effect of FG. Since STZ diabetes is an insulin deficient model, the probability of insulinomimetic effect seems more probable. Feeding of FG (2 g/kg) for 30 days caused a significant reduction in glucose levels by almost 50% though euglycemia was not achieved. In a recent study (Raju et al., 2001), complete normalization of blood glucose with feeding of 5% diet of powdered FG seeds was reported, a reduction that was not seen in the present study. This could be attributed to the presence of high fiber (>90%) and saponin content in the seeds of FG, which could have contributed partly to euglycemia in their study (Moorthy et al., 1989; Sauvaire et al., 1996; Buyken et al., 1999). Since we had used defatted extract (which is devoid of fiber content), it is possible that lack of fibers contributed to the lack of euglycemia. Moreover, in a previous study (Vats et al., 2002), we had shown that feeding single dose FG extract did not affect peak glucose levels in glucose-fed hyperglycemic rats implying that FG extract does not affect glucose absorption from the gut. STZ induced diabetes is characterized by severe loss in body weight (Chen and Ianuzzo, 1982; Raju et al., 2001) and this was also seen in the present study. While the normal rats registered approximately 30% growth in body weight diabetic rats showed no gain. FG prevented this loss in body weight significantly. However, it did not normalize the body weight completely as it remained significantly lesser than normal controls. Assessment of glycogen levels serves as a marker for studying insulinomimetic activity. Glycogen content of skeletal muscle and liver markedly decreases in diabetes and this alteration is normalized by insulin treatment (Prasannan
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and Subrahmanyam, 1965; Welihinda and Karunanayake, 1986; Grover et al., 2000). In the diabetic controls after 5 weeks of STZ administration, glycogen content in liver and muscle was reduced by approximately 75 and 68%, respectively, in comparison to the non-diabetic controls. This is in line with previous studies utilizing similar dose of STZ and carried for approximately same period (Khandelwal et al., 1977; Ferrannini et al., 1990) except from the study of Chen and Ianuzzo (1982) who reported lesser reduction in hepatic glycogen content though the duration and dose of diabetogenic agent was comparable to the present study. Treatment with FG for 30 days significantly increased the glycogen content by approximately 50% in muscle and 120% in the liver but lesser than the normal controls indicating that the defective glycogen storage of the diabetic state was only partially corrected by the herb. The glycogen content of insulin-dependent tissues is known to decrease in STZ diabetes but the opposite happens in insulin-independent tissues like kidneys (Belfiore et al., 1986). In the present study, renal glycogen content of diabetic rats was elevated by almost 13-folds in comparison to normal controls and is likely due to increased glycogen deposition. This is in agreement with earlier findings (Ross and Goldman, 1971; Spiro and Spiro, 1971). Feeding of FG failed to normalize renal glycogen deposition though it significantly reduced its levels. This may be due to failure in achieving euglycemia by the FG extract as glucose levels on the 30th day were still higher in comparison to the normal controls. In the present study, brain glycogen levels were not affected either in diabetic controls or in the treated groups and this in agreement with our earlier findings (Grover et al., 2000) though the duration of the work in the present case was longer. Diabetic rats had significantly higher liver weight/100 g body weight and this alteration in the diabetics was not altered by treatment with FG. Literature shows conflicting reports on the effect of diabetes on liver weight. While some report an increase in liver weight in animals (Murphy and Anderson, 1974; Chen and Ianuzzo, 1982; Sadique et al., 1987) as well as humans (Van Lancker, 1976), no change in liver weight and decrease (alloxan 200 mg/kg induced diabetes, study duration 21 days) has also been reported (Gupta et al., 1999; Raju et al., 2001). Exact reasons of hepatic hypertrophy are not known. However, fat deposition has been proposed to be the cause (Van Lancker, 1976). Several probable mechanisms have been suggested to explain the mechanism of action of FG. These include insulin secretion (Riyad et al., 1988; Khosla et al., 1995), insulinomimetic activity or inhibition of intestinal glucosidase (Petit et al., 1993). The current study provides some useful insight into the molecular effect of feeding FG. However, we suggest that further work should be carried out with whole seed powder and not their extracts. FG is widely distributed throughout the country and people commonly use leaves and seeds as an edible item implies a relative lack of toxicity. Moreover, leaves have also been
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shown to have an anti-hyperglycemic effect (Abdel-Barry et al., 1997, 2000). These findings suggest that consumption of FG seeds and leaves should be promoted as a constituent of diet in higher proportion in diabetic patients as well as those prone to getting diabetes.
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Riyad, M.A., Abdul-Salam, S.A., Mohammad, S.S., 1988. Effect of fenugreek and lupine seeds on the development of experimental diabetes in rats. Planta Medica 54, 286–290. Ross, J., Goldman, J.K., 1971. Effect of streptozotocin-induced diabetes on kidney weight and compensatory hypertrophy in the rat. Endocrinology 88, 1079–1082. Sadique, J., Begum, V.H., Thenmozhi, V., Elango, V., 1987. Hypoglycemic effect of cottonseed aqueous extract in alloxan induced diabetes mellitus in rats. Biochemical Medicine and Metabolic Biology 38, 104–110. Sauvaire, Y., Baissae, Y., Leconte, O., Petit, P., Ribes, G., 1996. Steroid saponins from fenugreek and some of their biological properties. Advances in Experimental Medicine and Biology 405, 37–46. Spiro, R.G., Spiro, M.J., 1971. Effect of diabetes on the biosynthesis of the renal glomerular basement membrane. Studies on the glucosyltransferase. Diabetes 20, 641–648. Steiner, D.F., King, J., 1964. Induced synthesis of hepatic uridine diphosphate glucose glycogen glycosyltransferase after administration of insulin to alloxan diabetic rats. Journal of Biology and Chemistry 239, 1292–1298. Swanston-Flatt, S.K., Day, C., Flatt, P.R., Gould, B.J., Bailey, C.J., 1989. Glycemic effect of traditional European plants treatments for diabetes. Studies in normal and streptozotocin diabetic mice. Diabetes Research 10, 69–73. Tan, A.W., Nuttall, F.Q., 1976. Regulation of synthase phosphatase and phosphorylase in rat liver. Biochimica et Biophysica Acta 445, 118– 130. Taylor, R., Agius, L., 1988. The biochemistry of diabetes. Biochemistry Journal 250, 650–740. Trinder, P., 1969. Determination of blood glucose using an oxidase–peroxidase system with a non-carcinogenic chromogen. Journal of Clinical Pathology 22, 158–161. Van Lancker, J.L., 1976. Molecular and Cellular Mechanism in Diseases. Springer Verlag, New York, USA, p. 497. Vats, V., Grover, J.K., Rathi, S.S., 2002. Evaluation of antihyperglycemic and hypoglycemic effect of Trigonella foenumgraecum, Ocimum sanctum and Pterocarpus marsupium in normal and alloxanized diabetic rats. Journal of Ethnopharmacology 79, 95–100. Weber, G., Lea, M.A., Fisher, E.A., Stamm, N.B., 1966. Regulatory pattern of liver carbohydrate metabolizing enzymes insulin as an inducer of key glycolytic enzymes. Enzymology Biology Clinics (Basel) 7, 11–24. Welihinda, J., Karunanayake, E.H., 1986. Extra-pancreatic effects of Momordica charantia in rats. Journal of Ethnopharmacology 17, 247–255. Whitton, P.D., Hems, D.A., 1975. Glycogen synthesis in the perfused liver of streptozotocin-diabetic rats. Biochemistry Journal 150, 153–165. Yajnik, C.S., 2001. The insulin resistance epidemic in India: fetal origins, later lifestyle, or both? Nutrition Reviews 59, 1–9.
Effect of T. foenumgraecum on glycogen content of tissues and the key enzymes of carbohydrate metabolism V. Vats, S.P. Yadav, J.K. Grover∗ Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110049, India Received 31 May 2002; received in revised form 20 December 2002; accepted 20 December 2002
Abstract The Indian traditional system of medicine prescribed plant therapies for diseases including diabetes mellitus called madhumeh in Sanskrit. One such plant mentioned in Ayurveda is Trigonella foenumgraecum (FG). In the present study, FG (1 g/kg PO) was assessed for its effect on glycogen levels of insulin dependent (skeletal muscle and liver), insulin independent tissues (kidneys and brain) and enzymes such as glucokinase (GK), hexokinase (HK), and phosphofructokinase (PFK). Administration of FG led to decrease in blood glucose levels by 14.4 and 46.64% on 15th and 30th day of the experiment. Liver and 2-kidney weight expressed as percentage of body weight was significantly increased in diabetics (P < 0.0005) versus normal controls and this alteration in the renal weight (P < 0.0005) but not liver weight was normalized by feeding of FG. Renal glycogen content increased by over 10 folds while hepatic and skeletal muscle glycogen content decreased by 75 and 68% in diabetic controls versus controls and these alteration in glycogen content was partly prevented by FG. Activity of HK, GK and PFK in diabetic controls was 35, 50 and 60% of the controls and FG partially corrected this alteration in PFK, HK and GK. © 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: T. foenumgraecum; Methi; Streptozotocin; Experimental diabetes; Carbohydrate metabolism
1. Introduction It is projected that incidence of diabetes is on rise. Present number of diabetics worldwide is 150 million and this is likely to increase to 300 million or more by the year 2025 (King et al., 1998). Reasons for this rise include increase in sedentary lifestyle, consumption of energy rich diet, obesity, higher life span, etc. (Yajnik, 2001). Though biguanides and sulfonylureas are valuable in treatment of diabetes mellitus, their use is restricted by their limited action, pharmacokinetic properties, secondary failure rates and accompanying side effects (Bailey et al., 1989). Moreover, these therapies only partially compensate for metabolic derangements seen in diabetics and do not necessarily correct the fundamental biochemical lesion (Taylor and Agius, 1988). Nature has been a source of medicinal treatments for thousands of years, and plants-based systems continue to play an essential role in the primary health care of 80% of the world’s underdeveloped and developing countries (King et al., 1998). ∗ Corresponding author. Tel.: +91-11-659-4897 (O)/461-5315 (R); fax: +91-11-686-2663. E-mail address: [email protected] (J.K. Grover).
Biguanides developed from a prototypic plant molecule is an excellent example of anti-diabetic drug development from plants. Thus, it is prudent in the current context to look for new and if possible more efficacious hits from the vast reserves of phytotherapy. Trigonella foenumgraecum Linn (FG) has been shown to possess hypoglycemic activity in experimental animals (Ghafghazi et al., 1977; Ribes et al., 1984, 1986; Swanston-Flatt et al., 1989; Ali et al., 1995; Khosla et al., 1995). However, very little is known about cellular and biochemical mechanism of the hypoglycemic or anti-hyperglycemic effect of FG. Although Raju et al. (2001) have shown that FG favorably effects glycolytic and gluconeogenic enzymes, they have worked with a whole seed powder diet. Since seeds have a very high fiber content, it is not possible to ascertain whether the anti-diabetic effect seen in their study was due to the effect of solid seed contents or some active pharmacological constituents. Therefore, this study was undertaken to assess the effect of FG on key enzymes of carbohydrate metabolism and glycogen content of various tissues. The dose was based on a previous pilot work that was undertaken to establish and assess the dose–response relationship (Vats et al., 2002).
0378-8741/03/$ – see front matter © 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00022-9
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2. Materials and methods
2.5. Biochemical and enzymatic estimations
2.1. Preliminary phytochemical evaluation
2.5.1. Plasma glucose Glucose levels were estimated by commercially available glucose kits based on glucose oxidase method (Trinder, 1969) (Autopak® , Bayer Diagnostics, Baroda).
It yielded moisture content of 10.3%, dry matter 89.7% (fiber 56.2%, protein 18.2%, mineral constituent 2.1%, alkaloidal content 0.011%, fixed oil in traces). The dose was 2 g/kg body weight. 2.2. Animals Male albino rats (150–200 g) were obtained from the experimental animal facility of the All India Institute of Medical Sciences. Before initiation and during the experiment, rats were fed standard chow diet. After randomization into various groups, the rats were acclimatized for 2–3 days in the new environment before initiation of experiment. Animals had free access to food and drinking water till before 30 min of sampling.
2.5.2. Hepatic phosphofructokinase activity (EC 2.7.1.11) The liver homogenate was prepared by grinding frozen tissue in ratio of 1:4 in 50 mM imidazole, 5 mM EDTA, 5 mM EGTA, 100 mM NaCl and 30 mM beta-mercaptoethanol (pH = 7). The homogenate was centrifuged at 12,000 × g for 15 min and supernatant was removed and stored in ice until assay. The phosphofructokinase activity was assayed spectrophotometrically by the method of Racker (1947). 2.5.3. Hepatic glucokinase activity The above-mentioned sample was used and enzyme activity was based on spectrophotometric method assaying glucose phosphorylation described by Pilkis (1975).
2.3. Experimental design All the animals were randomly divided into 3 groups with 10 animals in each group. Group I (CNT) were normal and used as controls. Group II (DCNT) were used as diabetic control. Group III was daily treated with FG after 10 days of standing hyperglycemia for 30 days. Streptozotocin (STZ) (Sigma, USA) was administered in Groups II–IV by a single intravenous injection of STZ (65 mg/kg) given intraperitoneally. STZ was first weighed in individual Eppendorf’s according to the weight of the animal, then solubilized with 0.2 ml saline (154 mM NaCl) just prior to injection. Rats exhibiting plasma glucose levels >300 mg/dl, 48 h after administration of STZ were included in the study. They were allowed a window period of 10 days before start of treatment. 2.4. Sample collection 2.4.1. Blood sample It was collected retro-orbitally from the inner canthus of the eye under light ether anesthesia using capillary tubes (Micro Hematocrit Capillaries, Mucaps). Blood was collected in fresh vials containing sodium fluoride and sodium oxalate as anti-coagulant/anti-glycolytic agents and plasma was separated in a T8 electric centrifuger (Remi Udyog, New Delhi) at 2000 rpm for 2 min. 2.4.2. Collection of organs After 30 days of daily feeding of FG orally (40 days after STZ), the animals were euthanized by overdose of intraperitoneal anesthesia and blood and tissue samples were collected for the assessment of plasma glucose, hepatic glucokinase (EC 2.7.1.1), hexokinase (EC 2.7.1.1), and phosphofructokinase (EC 2.7.1.11) content along with glycogen content in different tissues, i.e. liver, brain, heart and skeletal muscle.
2.5.4. Skeletal hexokinase activity The enzyme activity was determined by the method of Chou and Wilson (1975) and was based on the reduction of NADPH coupled with hexokinase measured spectrophotometrically at 340 nm. 2.5.5. Glycogen content The tissue sample was digested in hot concentrated 30% KOH, precipitated with ethanol, hydrolyzed and finally determined as glucose in the hydrolyzate as reducing sugar (Hassid and Abraham, 1957).
3. Results The basal levels of plasma glucose of the rats in all groups prior to STZ administration were not significantly different. However, 48 h after STZ administration, plasma glucose levels were significantly higher in the rats selected for the study. In contrast, non-diabetic controls remained persistently euglycemic throughout the course of the study. 3.1. Glucose levels The anti-hyperglycemic effect of FG on the blood sugar levels of diabetic rats is shown in Table 1. Controls rats did not show any significant variation in the blood glucose throughout the experimental period. Administration of STZ (65 mg/kg) led to over four-fold elevation of blood glucose levels (P < 0.001) which was maintained over a period of 5 weeks. After 4 weeks of daily treatment with FG led to a fall in blood sugar levels by 14.4 and 46.64% on 15th and 30th day of the experiment, respectively (P < 0.01 and P < 0.0001, respectively).
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Table 1 Effect of FG administration (30 days) on glucose levels (mg/%) in STZ (65 mg/kg) diabetic rats
CNT DCNT FG
0 day
10th day
25th day
40th day
78.76 ± 5.38 (n = 10) 77.90 ± 4.21 (n = 10) 80.87 ± 8.01 (n = 10)
81.29 ± 5.44 (n = 10) 325.43 ± 18.95∗ (n = 8) 322.86 ± 14.45 (n = 10)
82.11 ± 4.81 (n = 10) 318.28 ± 18.76∗ (n = 7) 276.21 ± 34.67∗ (n = 9) (14.44%)
84.39 ± 6.77 (n = 10) 321.53 ± 16.52∗ (n = 6) 172.27 ± 21.63∗∗ (n = 8) (46.64%)
CNT: control; DCNT: diabetic control. Values are given as mean ± S.D. for no. of animals indicated. Value in parenthesis indicates the percentage lowering of plasma sugar in comparison to the previous reading. Diabetic control was compared with normal control at the corresponding time interval and treated group was compared with the corresponding values at 48 h. ∗ Statistically significant at P < 0.01. ∗∗ Statistically significant at P < 0.0001.
Table 2 Effect of PM on body, liver and kidney weight in rats
Body weight on 0 day Body weight on 15th day Body weight on 30th day Liver weight on 30th day (g) Liver weight/100 g body weight on 30th day Kidneys weight on 30th day (g) Kidneys weight/100 g body weight on 30th day
CNT
DCNT
FG
163.1 ± 7.88 191.4 ± 9.14 213.2 ± 6.98 7.62 ± 0.68 3.53 ± 0.25 1.54 ± 0.08 0.71 ± 0.02
163.9 ± 10.98 165.7 ± 3.94∗∗∗ 168.7 ± 5.06∗∗∗ 7.45 ± 0.38 4.36 ± 0.16∗∗∗ 1.56 ± 0.04 0.92 ± 0.02∗∗∗
167.7 ± 13.6 175.4 ± 11∗ 189 ± 11∗∗,∗∗∗ 7.95 ± 0.59 4.18 ± 0.15 1.58 ± 0.09 0.84 ± 0.04∗∗
Abbreviations as for Table 1. Values are given as mean ± S.D. Value in parenthesis indicates the percentage increase or decrease vs. controls. Diabetic control was compared with normal control at the corresponding time interval and treated group was compared with diabetic controls. ∗ Statistically significant at P < 0.05. ∗∗ Statistically significant at P < 0.005. ∗∗∗ Statistically significant at P < 0.0005.
3.2. Body weight, renal and liver weight The effect of STZ and feeding FG on the body weight, renal and liver weight is shown in Table 2. There was no significant intra-group variation in the basal body weight on the 0 day of the experiment. While control rats gained significant weight in the 30-day period, there was no appreciable increase in the diabetic controls over the same period. On the other hand, treated rats gained significantly more weight than diabetic controls but the increase remained significantly lesser than the normal controls. There was no significant difference in the absolute weights of livers and kidneys among any of the experimental groups. However, liver and 2-kidney weight expressed as percentage of body weight was significantly increased in diabetics
(P < 0.0005) versus normal controls and this alteration in the renal weight but not liver weight was significantly reduced (P < 0.005) by feeding FG. 3.3. Glycogen content Glycogen content of various tissues (liver, skeletal muscle, heart, brain and kidneys) was estimated on the 30th day in normal controls, diabetic controls and FG-treated groups (see Table 3). As evident, the glycogen content decreased significantly by 75 and 68% in liver and skeletal muscle in diabetic controls as compared to non-diabetic controls. On the other hand renal glycogen content increased by over 10 folds in diabetic animals versus non-diabetic animals. Brain and heart glycogen content remained unaltered in diabetic
Table 3 Effect of FG administration (30 days) on glycogen content (mg/g tissue) of various tissues of STZ (65 mg/kg) diabetic rats Groups
Brain (mg/100 g)
Kidney (mg/g)
Heart (mg/g)
Muscle (mg/g)
Liver (mg/g)
CNT (n = 10) DCNT (n = 6) FG (n = 8)
40.34 ± 4.76 43.11 ± 6.41 40.33 ± 7.04
0.83 ± 0.22 11.5 ± 2.99∗∗ (↑1280%) 6.95 ± 1.48∗
1.81 ± 0.32 1.85 ± 0.58 1.8 ± 0.65
8.71 ± 1.22 2.81 ± 1.26∗∗ (↓68%) 4.6 ± 1.5∗
52.55 ± 5.22 13.25 ± 3.99∗∗ (↓75%) 30.15 ± 6.76∗∗
Abbreviations as for Table 1. Values are given as mean ± S.D. for no. of animals indicated. Value in parenthesis indicates the percentage increase or decrease vs. controls. Diabetic control was compared with normal control at the corresponding time interval and treated group was compared with diabetic control. ∗ Statistically significant at P < 0.005. ∗∗ Statistically significant at P < 0.0005.
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Table 4 Effect of administration (30 days) on levels of three important enzymes (%) involved in carbohydrate metabolism in STZ (65 mg/kg) diabetic rats Enzyme PFK GK HK
CNT (n = 10)
DCNT (n = 6)
FG (n = 8)
100 100 100
59.07∗∗∗
86.13∗∗ 73.20∗∗ 62.33∗∗
48.69∗∗ 35.56∗∗∗
Abbreviations as for Table 1. Values are given as mean ± S.D. for no. of animals indicated. Value in parenthesis indicates the percentage increase or decrease vs. controls. Diabetic control was compared with normal control at the corresponding time interval and treated group was compared with diabetic controls. ∗∗ Statistically significant at P < 0.001. ∗∗∗ Statistically significant at P < 0.0001.
animals while FG significantly attenuated the STZ-induced hepatic (P < 0.0005), and muscle (P < 0.005) renal (P < 0.005) glycogen content. 3.4. Hepatic enzymes The diabetic controls showed significant decrease in the values of PFK, GK and HK (see Table 4). The respective percentage decrease was approximately 40, 50 and 65% as compared to non-diabetic control values. As compared to diabetic controls, FG significantly altered these enzymes favorably but could not normalize them to the control values.
4. Discussion The present study was carried out to determine effect of feeding of FG on the key enzymes involved in carbohydrate metabolism in STZ (65 mg/kg) rats and glycogen content of insulin dependent (skeletal muscle and liver) and independent tissues (kidneys and brain). Diabetes mellitus is characterized by partial or total deficiency of insulin resulting in derangement of carbohydrate metabolism and a decrease in enzymatic activity of glucokinase, hexokinase and phosphofructokinase resulting in depletion of liver and muscle glycogen (Murphy and Anderson, 1974). GK and PFK are the most sensitive indicators of the glycolytic pathway in the diabetic state (Steiner and King, 1964). Since insulin administration normalizes these alterations in the enzymatic activities (Weber et al., 1966), these enzymes represent a method to assess peripheral utilization of glucose. In the present case, diabetic rats showed a significant reduction in all the three enzymes in comparison to the normal controls. Liver plays an important role in buffering the postprandial hyperglycemia and is involved in synthesis of glycogen. Diabetes mellitus is known to impair the normal capacity of the liver to synthesize glycogen (Hornbrook, 1970; Migliorini, 1971; Whitton and Hems, 1975). However, after food, diabetic animals fail to synthesize glycogen from glucose and gluconeogenic precursors. Synthase phosphatase activates glycogen synthase resulting in glycogenesis and
this activation appears to be defective in STZ-induced diabetic animals (Bishop, 1970; Tan and Nuttall, 1976; Golden et al., 1979). This inhibition of synthase phosphatase is almost complete after 3 days in alloxan-diabetic rats and in 1–2 weeks in STZ diabetes (Langdon and Curnow, 1983). Therefore, the drug treatment was started 10 days after STZ administration and continued for 4 weeks. STZ-induced diabetic animals tend to show renal hypertrophy. In the present case also, diabetic rats showed 29% increase in 2-kidney versus body weight ratio in comparison to controls. This degree of renal enlargement is lesser than 62 and 52% reported previously (Rasch, 1980; Nielsen et al., 1999). Probably degree of increase in renal weight is a time-dependent phenomenon as duration in the above-mentioned two studies was 6 and 2 months, respectively, as compared to 1 month in the present case. Forty days after STZ injection, GK and PFK levels of the diabetic rats were 48.69 and 59.07%, respectively of the controls and this is similar to previous finding (Steiner and King, 1964) in which both the dose of STZ and duration of diabetes is comparable with the present study. An increase in the activity of glucokinase and PFK in the FG-treated rats implies that cellular entry of glucose was facilitated by FG, which in turn stimulated the activity of these enzymes. This glucose influx could either be due to an insulin releasing or direct insulinomimetic effect of FG. Since STZ diabetes is an insulin deficient model, the probability of insulinomimetic effect seems more probable. Feeding of FG (2 g/kg) for 30 days caused a significant reduction in glucose levels by almost 50% though euglycemia was not achieved. In a recent study (Raju et al., 2001), complete normalization of blood glucose with feeding of 5% diet of powdered FG seeds was reported, a reduction that was not seen in the present study. This could be attributed to the presence of high fiber (>90%) and saponin content in the seeds of FG, which could have contributed partly to euglycemia in their study (Moorthy et al., 1989; Sauvaire et al., 1996; Buyken et al., 1999). Since we had used defatted extract (which is devoid of fiber content), it is possible that lack of fibers contributed to the lack of euglycemia. Moreover, in a previous study (Vats et al., 2002), we had shown that feeding single dose FG extract did not affect peak glucose levels in glucose-fed hyperglycemic rats implying that FG extract does not affect glucose absorption from the gut. STZ induced diabetes is characterized by severe loss in body weight (Chen and Ianuzzo, 1982; Raju et al., 2001) and this was also seen in the present study. While the normal rats registered approximately 30% growth in body weight diabetic rats showed no gain. FG prevented this loss in body weight significantly. However, it did not normalize the body weight completely as it remained significantly lesser than normal controls. Assessment of glycogen levels serves as a marker for studying insulinomimetic activity. Glycogen content of skeletal muscle and liver markedly decreases in diabetes and this alteration is normalized by insulin treatment (Prasannan
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and Subrahmanyam, 1965; Welihinda and Karunanayake, 1986; Grover et al., 2000). In the diabetic controls after 5 weeks of STZ administration, glycogen content in liver and muscle was reduced by approximately 75 and 68%, respectively, in comparison to the non-diabetic controls. This is in line with previous studies utilizing similar dose of STZ and carried for approximately same period (Khandelwal et al., 1977; Ferrannini et al., 1990) except from the study of Chen and Ianuzzo (1982) who reported lesser reduction in hepatic glycogen content though the duration and dose of diabetogenic agent was comparable to the present study. Treatment with FG for 30 days significantly increased the glycogen content by approximately 50% in muscle and 120% in the liver but lesser than the normal controls indicating that the defective glycogen storage of the diabetic state was only partially corrected by the herb. The glycogen content of insulin-dependent tissues is known to decrease in STZ diabetes but the opposite happens in insulin-independent tissues like kidneys (Belfiore et al., 1986). In the present study, renal glycogen content of diabetic rats was elevated by almost 13-folds in comparison to normal controls and is likely due to increased glycogen deposition. This is in agreement with earlier findings (Ross and Goldman, 1971; Spiro and Spiro, 1971). Feeding of FG failed to normalize renal glycogen deposition though it significantly reduced its levels. This may be due to failure in achieving euglycemia by the FG extract as glucose levels on the 30th day were still higher in comparison to the normal controls. In the present study, brain glycogen levels were not affected either in diabetic controls or in the treated groups and this in agreement with our earlier findings (Grover et al., 2000) though the duration of the work in the present case was longer. Diabetic rats had significantly higher liver weight/100 g body weight and this alteration in the diabetics was not altered by treatment with FG. Literature shows conflicting reports on the effect of diabetes on liver weight. While some report an increase in liver weight in animals (Murphy and Anderson, 1974; Chen and Ianuzzo, 1982; Sadique et al., 1987) as well as humans (Van Lancker, 1976), no change in liver weight and decrease (alloxan 200 mg/kg induced diabetes, study duration 21 days) has also been reported (Gupta et al., 1999; Raju et al., 2001). Exact reasons of hepatic hypertrophy are not known. However, fat deposition has been proposed to be the cause (Van Lancker, 1976). Several probable mechanisms have been suggested to explain the mechanism of action of FG. These include insulin secretion (Riyad et al., 1988; Khosla et al., 1995), insulinomimetic activity or inhibition of intestinal glucosidase (Petit et al., 1993). The current study provides some useful insight into the molecular effect of feeding FG. However, we suggest that further work should be carried out with whole seed powder and not their extracts. FG is widely distributed throughout the country and people commonly use leaves and seeds as an edible item implies a relative lack of toxicity. Moreover, leaves have also been
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shown to have an anti-hyperglycemic effect (Abdel-Barry et al., 1997, 2000). These findings suggest that consumption of FG seeds and leaves should be promoted as a constituent of diet in higher proportion in diabetic patients as well as those prone to getting diabetes.
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