Effect of Zinc-3 hydroxyflavone Complex on Carbohydrate Metabolic Enzymes in Hepatic and Renal Tissues of Streptozotocin Induced Experimental Diabetes in Rats

 

Kalavakunda Vijayaraghavan¹, Subramanian Iyyam Pillai² and Sorimuthu Pillai Subramanian¹*

¹Department of Biochemistry, University of Madras, Guindy campus, Chennai – 600 025, Tamil Nadu, India.

²Department of Inorganic Chemistry, University of Madras, Guindy campus, Chennai – 600 025, Tamil Nadu, India.

*Corresponding Author E-mail: subbus2020@yahoo.co.in

 

The exploitation of metals as therapeutic drugs becomes imperative over the last couple of decades resulting in a variety of exciting and valuable metallopharmaceutical drugs. Recently, we have designed, synthesized and characterized a novel zinc flavonol complex and its toxicity as well as antidiabetic efficacy was studied in streptozotocin-induced experimental diabetes in rats. In the present study the activities of key enzymes involved in carbohydrate metabolism were assayed to establish the effect of zinc flavonol complex in maintaining the glucose homeostasis. Oral treatment of zinc flavonol complex (5 mg/kg b.w/rat/day) to diabetic rats for 30 days resulted in a significant decline in blood glucose and glycosylated hemoglobin levels and a significant improvement in plasma insulin level. The altered activities of hexokinase, pyruvate kinase, glucose-6-phosphatase, fructose-1, 6-bisphosphatase, glucose-6-phosphate dehydrogenase, glycogen synthase and glycogen phosphorylase observed in liver and kidney tissues of diabetic rats were significantly reverted to near normalcy by the oral administration of zinc flavonol complex. The results of the present study indicate that the metallocomplex play a pivotal role in regulating key enzymes of carbohydrate metabolism to maintain normoglycemia.

 

 

 

INTRODUCTION:

Diabetes mellitus is an endocrine disorder resulting in persistant elevation of blood glucose under both fasting and postprandial conditions resulting in micro and macrovascular complications¹. The prevalence of diabetes is increasing globally and is predicted to increase by two-fold from 150 million in the year 2000 to 300 million by the year 2030². The abnormal regulation of glucose metabolism that results from a defective/deficient insulin secretion is the key pathogenic event in diabetes mellitus. Currently available drugs for normoglycemia exhibit adverse side effects on prolonged use. Hence the search for novel therapeutic drugs continues. In addition to drugs from natural sources, recent focus has been made in the synthesis of novel metallo complexes with pharmacologically important ligands, preferably from natural sources for the successful treatment of diabetes.

 

The development of various ligands in order to reduce the toxicity of zinc has increased in recent past to synthesize more effective and nontoxic zinc complex as insulin mimics³.

 

Flavonoids are a diverse group of polyphenolic phytoconstituents that are produced as secondary metabolites by green plants in appreciable amounts⁴. They exhibit a multitude of biological properties such as antioxidant, antibacterial, antiinflammatory, antiallergic, vasodilatory and anticarcinogenic⁵. Due to their abundance in dietary products and potential pharmacological and nutritional benefits, flavonoids are of considerable interest for drug development as well as health food supplements. Flavonoids have also been recognized as the active components in a number of traditional ethnic remedies for diabetes⁶. Among the flavonoids, flavonol is known to chelate metal ions with great affinity owing to the presence of a a hydroxy-carbonyl group⁷. Based on this property, flavonol was chosen as a ligand for the synthesis of zinc complex in the study. More recently, we have established the non toxic and antidiabetic efficacy of the zinc flavonol complex in streptozotocin-induced experimental diabetes in rats.

 

Liver is a major site of endogenous glucose production with a minor contribution to kidney, produces glucose by glycogenolysis and gluconeogenesis. Numerous studies have provided influential evidence that hepatic glucose production (HGP) plays an important role in the development of fasting hyperglycemia in diabetes. The enzymes that regulates hepatic glucose metabolism are potential targets for controlling endogenous glucose production and thereby blood glucose levels in diabetes. Hence, the present study was designed to evaluate the regulatory effect of zinc flavonol complex on glucose metabolism in hepatic and renal tissues in STZ induced experimental diabetes in rats.

 

MATERIALS AND METHODS

Synthesis of Zinc-flavonol complex

Molar ratio method was followed in the synthesis of Zn-flavanol complex. Because of the very low solubility of these compounds in water, spectrophotometric graded ethanol was used. The zinc-flavonol complex was synthesized as previously reported with slight modifications⁷. Briefly, an ethanolic solution containing zinc acetate dihydrate (0.2195g, 1mM) was gradually added to an equimolar ethanolic solution of flavonol (0.2382g, 1mM).  The pH of the medium was adjusted to 7.5 with Tris-HCl buffer and the reaction mixture was constantly stirred, refluxed for 4 hours at 80^oC over an oil bath⁸. The resulting precipitate was filtered, washed with absolute ethanol, dried in vacuum and a yellow colour zinc flavonol (yield of 98%) was obtained. The metallo complex was characterized and used for further studies. 

 

Experimental Animals

Male albino rats of Wistar strain weighing (160-180g) were procured from Tamilnadu Veterinary and Animal Sciences University (TANUVAS), Chennai. The rats were housed in spacious polypropylene cages lined with husk. The experimental rats were maintained in a controlled environment (12:12 ± 1h light/dark cycle) and temperature (30°C ± 2).  Animals were acclimatized to standard husbandry conditions for one week to eliminate the effect of stress prior to initiation of the experiments. The rats were fed with commercial pelleted rats chow (Hindustan Lever Ltd., Bangalore, India), and had free access to water ad libitum. The experiments were designed and conducted in strict accordance with the current ethical norms approved by Ministry of Social justices and Environment, Government of India and Institutional Animal Ethical Committee guidelines [IAEC NO: 01/060/09].

 

Induction of experimental diabetes

Rats were fasted overnight and diabetes was experimentally induced by intraperitoneal injection of streptozotocin with a single dose of 50 mg/kg b.w/rat. Streptozotocin was dissolved in freshly prepared 0.1 M cold citrate buffer pH 4.5⁹. Since streptozotocin is capable of inducing fatal hypoglycemia as a result of massive pancreatic insulin release, streptozotocin-treated rats were provided with 10% glucose solution after 6 h for the next 24 h to prevent diabetogen induced hypoglycemia¹⁰. On 3^rd day the development and aggravation of diabetes in rats was confirmed and rats with fasting blood glucose concentration more than 250 mg/dL were selected for the experiments.

 

Experimental design

The animals were divided into four groups, comprising a minimum of six animals in each group as follows:

Group 1 - Control rats.

Group 2 - Streptozotocin induced diabetic rats.

Group 3 - Diabetic rats treated with zinc-flavonol (5 mg/kg b.w/rat/day) as aqueous suspension orally for 30 days.

Group 4 - Diabetic rats treated with gliclazide (5 mg /Kg. b.w/rat/day) in aqueous solution orally for 30 days. 

Biochemical estimations

Blood glucose and glycosylated hemoglobin were estimated according to the methods of Trinder¹¹, Nayak and Pattabiraman¹², respectively. Insulin level was measured in plasma using the sensitive rat insulin ELISA kit (Linco Research, Inc., St. Charles, MO). A portion of the liver and kidney tissues were dissected and washed immediately with ice-cold saline and were homogenized in 0.1M Tris–HCl buffer (pH 7.4) for the assay of key enzymes of carbohydrate metabolism. The homogenate was centrifuged at 10,000 rpm to remove the debris and the supernatant was used as enzyme source for the assays of hexokinase¹³, pyruvate kinase¹⁴, glucose-6-phosphatase¹⁵, fructose-1, 6-bisphosphatase¹⁶, glucose-6-phosphate dehydrogenase¹⁷, glycogen synthase¹⁸, glycogen phosphorylase¹⁹. Another portion of wet liver tissue was used for the estimation of glycogen content²⁰.

 

Statistical analysis

The results were expressed as mean ± S.E.M of six rats per group and statistical significance was evaluated by one-way analysis of variance (ANOVA) using SPSS (version 16.0) program followed by LSD. Values were considered statistically significant when P< 0.05.

 

RESULTS

Figure 1 represents the scheme of the synthesis of zinc flavonol complex. The zinc-flavonol complex (C₁₅H₉O₃Zn) obtained was yellow in colour and the yield was 98%.

 

 

Figure-1. Synthesis of Zn-flavonol complex.

 

Table 1. Effect of zinc flavonol complex on the levels of blood glucose, plasma insulin and glycosylated hemoglobin (HbA1c) in experimental groups of rats after 30 days experimental period

Units: Glucose – (mg/dl); Insulin – (ng/ml); HbA1c – (% Hemoglobin).Values are given as mean ± S.E.M for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the groups as follows: ^acontrol rats; ^bdiabetic control rats; Values are statistically significant at P < 0.05.

 

Table 2. Effect of zinc flavonol complex on the activities of hexokinase and pyruvate kinase in liver tissues of control and experimental groups of rats

Units are expressed as: µ moles of glucose-6-phosphate formed/h/mg of protein for hexokinase, mU/mg of protein for pyruvate kinase.Values are given as mean ± S.E.M for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the groups as follows: ^acontrol rats; ^bdiabetic control rats; Values are statistically significant at P < 0.05

 

Table 3. Effect of zinc flavonol complex on the activities of hexokinase and pyruvate kinase in kidney tissues of control and experimental groups of rats

Units are expressed as: µ moles of glucose-6-phosphate formed/h/mg of protein for hexokinase, mU/mg of protein for pyruvate kinase.Values are given as mean ± S.E.M. for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the groups as follows: ^acontrol rats; ^bdiabetic control rats;Values are statistically significant at P < 0.05.

 

Table 4: Activities of glucose-6-phosphatase, fructose-1, 6-bisphosphatase and glucose-6-phosphate dehydrogenase in liver tissues of control and experimental groups of rats

Units are expressed as: µmoles of Pi liberated/h/mg of protein for glucose-6-phosphatase and fructose-1,6-bisphosphatase and µmoles of NADPH/min/mg of protein for glucose-6-phosphate dehydrogenase. Values are given as mean ± S.E.M. for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the groups as follows: ^acontrol rats; ^bdiabetic control rats; Values are statistically significant at P < 0.05.

 

Table 5: Activities of glucose-6-phosphatase, fructose-1, 6-bisphosphatase and glucose-6-phosphate dehydrogenase in kidney tissues of control and experimental groups of rats

Units are expressed as: µmoles of Pi liberated/h/mg of protein for glucose-6-phosphatase and fructose-1, 6-bisphosphatase and µmoles of NADPH/min/mg of protein for glucose-6-phosphate dehydrogenase. Values are given as mean S.E.M. for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the groups as follows: ^acontrol rats; ^bdiabetic control rats; Values are statistically significant at p < 0.05.

 

Table 6: Level of glycogen content and activities of glycogen synthase and glycogen phosphorylase in liver tissues of control and experimental groups of rats

Units are expressed as: mg/g wet tissue for glycogen, µ moles of UDP formed/h/mg protein for glycogen synthase and µmoles Pi liberated/h/mg protein for glycogen phosphorylase. Values are given as mean ± S.E.M. for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the groups as follows: ^acontrol rats; ^bdiabetic control rats; Values are statistically significant at P < 0.05

 

 

Table 1 depicts the effect of oral administration of zinc flavonol on blood glucose, glycosylated hemoglobin and plasma insulin levels in experimental groups of rats. There was a significant elevation in the levels of blood glucose and glycosylated hemoglobin of streptozotocin induced diabetic rats as compared with control group of rats.Upon treatment with zinc flavonol as well as gliclazide for 30 days, diabetic rats showed a significant decrease in the levels of blood glucose and glycosylated hemoglobin, which were comparable with control group of rats. Moreover, the significantly diminished plasma insulin level of diabetic rats was improved significantly to near normal level by the administration with zinc flavonol as well as gliclazide.

 

Tables 2 and 3 depict the effect of zinc flavonol supplementation on the activities of hexokinase, pyruvate kinase in liver and kidney tissues of control and experimental groups of rats. The activities of hexokinase and pyruvate kinase were significantly diminished in liver and kidney tissues of streptozotocin induced diabetic rats. Oral administration of zinc flavonol to diabetic rats altered the activities of these to near normalcy in liver and kidney tissues similar to gliclazide treated rats

 

Table 4 and 5 depicts the activities of glucose-6-phosphatase, fructose-1, 6-bisphosphatase and glucose-6-phosphate dehydrogenase in liver and kidney tissues of control and experimental groups of rats. The liver and kidney tissues of diabetic rats showed a significant elevation in the activities of glucose-6-phosphatase and fructose-1, 6-bisphosphatase and a significant decrease in the activity of glucose-6-phosphate dehydrogenase. The altered activities of these enzymes were reverted to near normalcy by treatment with zinc flavonol as well as gliclazide in diabetic groups of rats

 

Table 6 represents levels of glycogen and the activities of glycogen synthase and glycogen phosphorylase in liver of control and experimental groups of rats. 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 group of rats. Oral treatment with zinc flavonol as well as gliclazide to diabetic rats restored the level of glycogen and the activities of glycogen synthase, glycogen phosphorylase to near normalcy when compared to control group of rats.

 

DISCUSSION:   

Zinc complexes so far evaluated for assessing antidiabetic potential were primarily aimed to facilitate better zinc absorption when compared to zinc salts, due to the lipophilic nature of the ligands. Zinc- flavonol complex is of significant clinical interest in the study of enhancing the presentation of zinc in the biological system. Earlier reports have revealed that oral administration of bis (allixinato) zinc complex at a dosage of 3mg/kg b.w/day to KKA^y mice improved glucose intolerance²¹. Zinc complexes exhibiting hypoglycemic potential and insulin mimetic activity have been previously reported.

 

Insulin regulates uptake and utilization of glucose in target organs such as liver, kidney, skeletal muscle and adipose tissue by controlling the activities of various metabolic enzymes. Zinc is found to be a part of larger number of enzymes which regulates carbohydrate and lipid metabolism²². Ezaki et al²³ demonstrated zinc mediated translocation of GLUT4 from cytosol to plasma membrane resulting in increased glucose uptake by tissues thereby facilitating normoglycemia. The glycemic status of the diabetic rats treated with zinc-flavonol complex exemplifies the effective role in maintaining glucose homeostasis. The observed increase in plasma insulin level in zinc flavonol treated rats demonstrates that the complex exerts its tissue protective nature on pancreas as well as stimulation of insulin secretion from remnant pancreatic βcells.

 

Glycosylated haemoglobin (HbA1c) is the clinical marker of chronic glycemic control in patients with diabetes mellitus²⁴. Persistent hyperglycemia leads to the glycosylation of amino groups of lysine residue in proteins²⁵. This condition favors reduction in the level of total hemoglobin and elevation in glycosylated hemoglobin, which in turn directly proportional to blood glucose²⁶. Diabetic rats showed higher levels of glycosylated hemoglobin indicating their poor glycemic control. The zinc-flavonol treatment to diabetic rats significantly reduced the HbA1c levels suggesting the ameliorative potential of the complex during hyperglycemia.

 

Liver plays a central role in the maintenance of glucose homeostasis²⁷. The uncontrolled hepatic glycogenolysis and gluconeogenesis and decreased utilization of glucose by the tissues are the fundamental factors contributing to a condition termed as hyperglycemia in diabetes mellitus²⁸. Hyperglycemic status occurs due to the lack of suppression of hepatic glucose production in the absorptive state and excessive glucose production in the post absorptive state. The enzymes that are involved in the regulation of hepatic glucose production are potential targets for controlling the glucose homeostasis in diabetes. Hence the present study was focused in assessing the activities of hepatic key enzymes of carbohydrate metabolism in STZ induced diabetic rats.

 

Hexokinase is a major regulatory enzyme involved in the oxidation of glucose. Since it is an insulin-dependent enzyme, the hepatic hexokinase activity in diabetic rats is almost entirely inhibited or inactivated due to the absence of insulin²⁹. This impairment results in a marked decline in the rate of glucose oxidation via glycolysis, which ultimately leads to hyperglycemia. The markedly decreased level of insulin observed in the streptozotocin-induced diabetic animals ultimately leads to the impairment in the activity of hexokinase, since insulin deficiency is a clinical hallmark of diabetes³⁰. Oral administration of zinc flavonol complex to streptozotocin-induced diabetic rats resulted in a significant reversal in the activity of hexokinase. The decreased fasting blood glucose levels observed in zinc flavonol complex administered diabetic rats may be due to increased hexokinase activity in the hepatic and renal tissues thereby increasing the oxidation of glucose leading to controlled glucose homeostasis.

 

Pyruvate kinase is a ubiquitously expressed key glycolytic enzyme that catalyzes the conversion of phosphoenol pyruvate to pyruvate with the generation of ATP and the altered expression could be expected to impair the glucose metabolism and energy production.  Pyruvate kinase is regulated by its own substrate phosphoenolpyruvate and fructose-1, 6-biphosphate an intermediate in glycolysis which both up-regulate pyruvate kinase. The observed decrease in the activity of PK in the liver and kidney of STZ induced diabetic rats readily accounts for the decreased utilization of glucose (glycolysis) and increased production of glucose (gluconeogenesis) by liver and kidney indicating that these two pathways are altered in diabetes³¹. Oral administration of zinc flavonol complex to streptozotocin- induced diabetic rats resulted in a significant increase in the activity of pyruvate kinase. The improved activities of hexokinase and pyruvate kinase suggest the effective utilization of glucose.

 

Glucose-6-phosphatase, a gluconeogenic enzyme, catalyzes the dephosphorylation of glucose-6- phosphate to glucose³². Fructose-1, 6- bisphosphatase is another gluconeogenic enzyme that catalyzes the dephosphorylation of fructose-1,6- bisphosphate to fructose-6-phosphate serves as a site for the regulation of gluconeogenesis³³. The increased activities of glucose 6-phosphatase and fructose-1,6-diphosphatase in liver and kidney of the STZ induced diabetic rats may be due to insulin insufficiency. Upon treatment with the zinc flavonol complex the activities of glucose-6-phosphatase, fructose-1, 6-diphosphatase were found to be decreased. This might be due to increased insulin secretion, which is responsible for the repression of the gluconeogenic key enzymes.

 

Glucose-6-phosphate dehydrogenase is the first rate limiting enzyme of the pentose phosphate pathway³⁴. The activity of glucose-6-phosphate dehydrogenase is found to be decreased in diabetic conditions³⁵. Oral treatment of zinc flavonol complex to streptozotocin-induced diabetic rats significantly increased the activity of glucose-6-phosphate dehydrogenase. It seems to increase the influx of glucose into the pentose monophosphate shunt in an effort to decrease high blood glucose level.

 

Glycogen is a branched polymer of glucose residues (the primary intracellular storable form of glucose) which is synthesized by the enzyme glycogen synthase³⁶. The two key regulatory enzymes that catalyze glycogenesis and glycogenolysis are glycogen synthase and glycogen phosphorylase. Zinc acts as an inhibitor of glycogen synthase kinase-3β (GSK-3β) thereby promoting glycogen synthase activity which adds to better disposal of glycemia. Glycogen synthase is the rate-limiting enzyme in glycogen metabolism which catalyzes the transfer of glucose from UDP-glucose to glycogen in animal cells. Because of its central role in glucose homeostasis, glycogen synthase is responsive to endocrine factors, including insulin, glucagon, and catecholamines, as well as to metabolic status, such as the concentration of the allosteric activator glucose 6-phosphate (G6P). Further, the decreased glycogen content in diabetic condition is due to the increased activity of glycogen phosphorylase and decreased activity of glycogen synthase³⁷.

 

Glycogen phosphorylase, a rate-limiting enzyme of glycogenolysis, cleaves α (1→4) linkage to remove glucose molecules from the glycogen. During diabetic conditions, the glycogen levels, glycogen synthase activity and responsiveness to insulin signaling are diminished and glycogen phosphorylase activity is significantly increased³⁸. Oral administration of zinc flavonol complex to diabetic rats regulated the activity of glycogen metabolizing enzymes thereby normalized the altered glycogen content.

 

Thus, the oral administration of zinc flavonol to STZ induced diabetic rats brings about the normoglycemia by regulating the key enzymes of carbohydrate metabolism. However, detailed studies involving the expression of these key enzymes are warranted to elucidate the exact mechanism of action of the zinc flavonol complex in controlling the hyperglycemia.

 

CONCLUSION:

The results of the study exemplifies that oral administration of zinc flavonol complex produce marked blood glucose-lowering effect in STZ diabetic rats by enhancing the effective utilization of glucose by the tissues. The improved activity of glycogen synthase reveals the improved glycogen content in the liver. It may be postulated that the zinc flavonol complex commits the system to glycolytic pathway by exerting its insulin mimetic action. Hence, zinc flavonol complex with its insulin mimetic action may be considered as a potential candidate for the successful treatment of diabetes mellitus.

 

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Accepted on 18.03.2012         © AJRC All right reserved