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Thioflavin T effect in diabetic Wistar rats: Reporting the antihyperglycemic property of an amyloid probing dye
Pharmacological Reports 67 (2015) 364–369
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Thioflavin T effect in diabetic Wistar rats: Reporting the antihyperglycemic property of an amyloid probing dye Mahmood Najafian a, Shahab Amini b,1, Babak Dehestani c,1, Kazem Parivar d, Azadeh Ebrahim-Habibi e,c,* a
Department of Biology, Jahrom Branch, Islamic Azad University, Jahrom, Iran Diabetes Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran d Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran e Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran b c
A R T I C L E I N F O
Article history: Received 10 August 2014 Received in revised form 18 October 2014 Accepted 20 October 2014 Available online 5 November 2014 Keywords: Thioflavin T Diabetes Streptozotocin Amyloid Amylase.
A B S T R A C T
Background: Thioflavin T (ThT) is a well-known probe of amyloid fibrils with a benzothiazole core structure. As a compound with partial inhibitory effect on alpha-amylase, the results of oral ThT administration were investigated on a streptozotocin (STZ)-induced rat model of diabetes. Methods: STZ was administered intraperitoneally for induction of diabetes. Afterwards, doses of 2, 8, 16, and 32 mg/kg of ThT were used in diabetic and non-diabetic rats. Blood glucose levels, lipid profiles, alpha-amylase activity, food and water intake and urine volume were assessed. Docking was also performed to evaluate the inhibitory effect of ThT on alpha-amylase. Results: Upon treatment with ThT, blood glucose levels and lipid profile of diabetic rats improved significantly. Furthermore, alpha-amylase serum levels of treated animals decreased compared to the control group, suggesting a possible effect of ThT on this digestive enzyme. On the other hand, the food intake of treated animals showed a decrease. ThT effects were also seen to some extent in the nondiabetic group. Conclusion: ThT is suggested to be a potentially useful compound in treatment and prevention of diabetes and associated complications. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Diabetes is one of the leading causes of mortality and morbidity in the modern world and reported to be among top causes of death in the recent years [1,2]. According to the statistics of International Diabetes Federation (IDF), the predicted prevalence of diabetes suggests that approximately 592 million people will suffer from diabetes by 2030 [3]. Furthermore, as a chronic disease, diabetes is accompanied with serious kinds of complications such as vasculopathy, nephropathy, neuropathy and retinopathy. These realities show the vital importance of efficient therapeutic methods [4–6]. Conventional
* Corresponding author. E-mail addresses: [email protected], [email protected] (A. Ebrahim-Habibi). 1 These authors have contributed equally to the work.
hypoglycemic agents usually focus on one specific pathway to fight against diabetes, while as a multi-factorial illness it would be preferable that a drug affected multiple targets simultaneously [4,7,8]. Besides, diabetic patients have to tolerate life-long medication with important side effects such as hypoglycemia, hepatocellular injury, neurological deficit, diarrhea, nausea, vomiting, flatulence while they may finally suffer from lack of appropriate response to conventional hypoglycemic agents in the long term [5,8,9]. Therefore, there is a growing necessity to find new antidiabetic drugs with the aid of well-planned research studies [6,8,9]. In this study, we focused on the characteristics of a small molecule named Thioflavin T (ThT). ThT is a benzothiazole dye with IUPAC (International Union of Pure and Applied Chemistry nomenclature) name of 4-(3,6-dimethyl-1,3-benzothiazol-3-ium2-yl)-N,N-dimethylaniline chloride [10,11]. It has been widely used as a defining probe to detect amyloid fibrils, based on its distinctive property of displaying dramatically enhanced fluorescence in its interaction with amyloid fibril, since 1959 [10–13].
http://dx.doi.org/10.1016/j.pharep.2014.10.013 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
M. Najafian et al. / Pharmacological Reports 67 (2015) 364–369
Among latest strategies of controlling diabetes, inhibition of mammalian alpha-amylase is recently considered as a therapeutic plan for the disease by lessening postprandial hyperglycemia [14–17]. Our research paradigm was based upon the hypothesis that, with regard to its structure, ThT may be a possible human alphaamylase inhibitor. Thus, we did docking studies to investigate the interaction between alpha-amylase and ThT and also evaluated the remedial effects of ThT’s administration in rats as an in vivo model.
Materials and methods Chemicals THT, dimethyl sulfoxide, soluble starch and maltose were obtained from Merck (Darmstadt, Germany). Streptozotocin, porcine pancreatic alpha-amylase (PPA) (E.C.3.2.1.1) and dinitrosalycilic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Animals All rats were male Wistar, weighting 200 15 g and 2.5 months age. They were kept in cages in groups of six, inside a room at temperature of 22–24 8C. During daylight hours (8:00 to 20:00), room lights were turned on. Following standard rodent diet was used: maintenance diet Letica, Panlab S.L., Barcelona, Spain; 61.4% (w/w) carbohydrate (100% starch), 3.9% fiber, 15.1% protein and 2.7% fat, and tap water. Rats were given ad libitum access to food and water. Induction of diabetic condition was performed by means of a single-dose streptozotocin’s (STZ) administration (70 mg/kg body weight) dissolved in a citrate buffer (0.1 mol/l), pH 4.5. The administration of STZ was intraperitoneal and was performed according to existing protocols as used in previously published related work [16]. Afterwards, we measured blood glucose concentration every 2 days. Rats were divided into two major groups, ‘‘non-diabetic rats’’ (ND) and ‘‘diabetic rats’’ (D), and each was subsequently divided into five subgroups, as defined below: non-diabetic control group (NDC) was given ligand solvent, distilled water (O) orally during 24 days with the use of a gastric cannula in single doses of 0.5 ml at 8:30 AM. Diabetic control group (DC) was administered with the use of a gastric cannula in single doses of 0.5 ml at 8:30 AM, during 24 days. Non-diabetic group receiving ThT (NDTh): ThT was administered orally at 2, 8, 16 and 32 mg/kg (groups were named NDTh2, NDTh8, NDTh16 and NDTh32, respectively): the compound was dissolved in distilled water, and given during 24 days with the use of a gastric cannula in single doses of 0.5 ml at 8:30 AM. Diabetic groups receiving ThT (DTh): ThT was administered at 2, 8, 16 and 32 mg/kg body weight (respectively DTh2, DTh8, DTh16 and DTh32) dissolved in distilled water, orally, during 24 days through a gastric cannula in single doses of 0.5 ml at 8:30 AM. Since this project was, to our knowledge, the first animal experiment on anti-diabetic therapeutic effects of ThT, the doses were chosen based on our in vitro experiences with ThT, as well as considering our previous study on trans-chalcone whose in vitro and in vivo effects were both studied as a potential hypoglycemic agent [16,18]. The ethical aspect of the experimental protocol was given ethical clearance approval (code: E-0033) by the ethics committee of the Endocrinology and Metabolism Research Center as founder of the project, and the ethical clearance was also approved by the internal ethical committee of the Science and Research Branch of Islamic Azad University, Tehran, where the research was conducted.
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Measured parameters Food and water intake and urine volume were assessed and quantified every morning at 9:00 AM. Blood glucose levels were measured in blood samples that were obtained from the tail of the animal every 2 days on mornings at 9:00 AM with a glucometer (One Touch Profile, Life Scan). Body weight of the rats was measured after day 24, and the animals were then sacrificed under light ether anesthesia. Blood samples collected from rat hearts were placed on ice and centrifuged within 15 min after blood collection at 3000 g for 5 min. The concentrations of cholesterol, triacylglycerol, high-density lipoproteins (HDL), low-density lipoproteins (LDL) and alpha-amylase activity were measured with standard biochemical kits at the end of our experiment (day 24). Estimates of very-low-density lipoprotein (VLDL) were calculated from the formula VLDL-C = triacylglycerol/5 [13]. Statistical analysis The data were analyzed by means of one-sample Kolmogrov– Smirnov test and Levene’s test. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons were used to compare difference between experimental groups. The criterion for statistical significant was p < 0.05. Enzyme inhibition experiment The process of enzyme assay was conducted by use of the Bernfeld method, as previously described [18]. Soluble starch was the substrate and the activity of alpha-amylase was defined as one unit being the amount of enzyme able to release 1 mmol of maltose from starch per minute. Enzyme remaining activity in the presence of different concentrations of thioflavin was calculated as a percentage by comparison with the control sample activity. Docking Docking was performed with AutodockVina and use of the Gasteiger charges for the receptor and ligand [19]. Receptor was the 1DHK.pdb file which had been processed with the use of MOE 2010.10 (Chemical Computing Group Inc., Montreal, Canada), and the protonation state of the structure was adjusted for neutral pH. A Grid box of 40 40 40 points was used with a spacing 1.0 A˚, with the grid box center was put on x = 102.186, y = 40.132 and z = 18.012. Preparation of the image representing the best pose was done with MOE 2010.10.
Results Glucose During the whole experience, the consequence of induction of diabetes in diabetic control groups was a notable increase in the level of blood glucose (408.6 3.8) compared with non-diabetic group (107.9 4.1). Treatment with ThT resulted into a significant subside in the level of glucose concentrations in diabetic rats (p < 0.05). As shown in Fig. 1, in all groups, this decrease was steep until the 6th day of administration of thioflavin, followed by slight decrease in all doses by 332.2, 273.1, 250.3 and 237.6 mg/ml in DTh2 DTh8, DTh16 and DTh32 groups, respectively. A similar trend was seen in non-diabetic groups. However, there were no significant differences between NDTh8, NDTh16 and NDTh32 (Fig. 2).
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Fig. 1. Blood glucose levels in the diabetic group (D); DO was the control group and 2, 8, 16 and 32 mg/kg ThT was administered to the treated groups.
Lipids There was a significant increase in the level of all lipid profiles in diabetic control groups (50.9, 71.9, 70.6 and 80.5% in cholesterol, TG, VLDL and LDL, in order), while HDL decreased 39.2%. Administration of ThT in different doses resulted in decrease in all lipids’ concentrations (Figs. 3 and 4). Although there were no significant differences between DTh8, DTh16 and DTh32 groups, these three groups showed a dramatic (p < 0.05) decrease in cholesterol and triglyceride levels in comparison with the first two groups both in non-diabetic and diabetic groups. Furthermore, by administration of 16 mg/kg ThT, HDL concentration recovered to normal level of 41.6 after the experience (p < 0.05). In non-diabetic groups, there were no noteworthy changes in VLDL, LDL and HDL (p > 0.05). To sum up, the ideal dose of ThT administration for correcting lipid profile was found to be 8 mg/kg.
Fig. 3. Lipid profile of the diabetic group; 8, 16 and 32 mg/kg ThT was administered to the treated groups. a: significantly different from diabetic control group!cholesterol; b: significantly different from diabetic Th2 group!cholesterol; g: significantly different from diabetic control group!triglyceride.
Alpha-amylase As illustrated in Fig. 5, in both diabetic and non-diabetic groups, serum alpha-amylase levels were decreased. The maximum inhibition was observed in DTh8, DTh16 and DTh32 (p < 0.05) in diabetics and in NDTh8, NDTh16 and NDTh32 in non-diabetic groups (p < 0.05). Changes in DTh8, DTh16 and DTh32 groups were significantly different from DTh2 and control group (p < 0.05).
Fig. 2. Blood glucose levels in the non-diabetic group (ND); NDO was the control group and 2, 8, 16 and 32 mg/kg ThT was administered to the treated groups.
Fig. 4. Lipid profile of the non-diabetic group; 8, 16 and 32 mg/kg ThT was administered to the treated groups.
Fig. 5. Alpha-amylase serum levels in diabetic and non-diabetic groups, 8, 16 and 32 mg/kg ThT was administered to the treated groups. Exponential trend line is also indicated. a: significantly different from non-diabetic control group; b: significantly different from non-diabetic Th2; a (italic): significantly different from diabetic control group; b (italic): significantly different from diabetic Th2; g (italic): significantly different from Th16.
M. Najafian et al. / Pharmacological Reports 67 (2015) 364–369
Fig. 6. Body weights of diabetic and non-diabetic rats, 8, 16 and 32 mg/kg ThT was administered to the treated groups. a: significantly different from diabetic control group; b: significantly different from diabetic Th2 group.
Body weight and food intake
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Fig. 8. Urine volume in diabetic and non-diabetic rats, 8, 16 and 32 mg/kg ThT was administered to the treated groups. a: significantly different from diabetic control group; b: significantly different from diabetic Th2 group; g: significantly different from diabetic Th8 group.
Use of thioflavin probably deemed to have appetite-suppressing property in both diabetic and non-diabetic groups. Additionally, as can be seen in Figs. 6 and 7, weight as an associated factor with food intake was decreased significantly in all treated groups compared with control group (p < 0.05). However, the minimum food intake among diabetic groups was noticed in DTh8, DTh16 and DTh32 groups. Higher food consumption of diabetic groups compared with non-diabetics would be a characteristic of the disease state. Urine and water intake As a consequence of being in diabetic states, diabetic rats tend to drink more water and urinate more compared with the other group. Figs. 8 and 9 depict the amount of water consumed by rats. Diabetic control group consumed 110.5 ml/day which is approximately three times more than non-diabetic control group. After administration of ThT, significant decrease in both parameters was seen.
Fig. 9. Water intake in diabetic and non-diabetic rats, 8, 16 and 32 mg/kg ThT was administered to the treated groups.
Probing the interaction of ThT with alpha-amylase In silico docking experiment showed that thioflavin T is able to interact with the residues located in the binding site cleft of mammalian alpha-amylase. The docking results provided
56 poses whose energies ranged from 6.1 to 3.5 kcal/mol. The best two poses (with lowest energies) are shown in Fig. 10. The two positioning differ from each other with regard to the location of the benzothiazole ring, but show similar conformations. In the absence of an amylase crystal structure containing thioflavin T, both these orientations seem plausible. Upon testing ThT inhibitory activity toward a mammalian alphaamylase in vitro, primary results were the observation of a partial inhibitory effect, with a maximal inhibition of 80% with the use of a 400 micromolar concentration. Further experiments showed a lower inhibitory potential, which seemed to have a non dosedependent pattern (results not shown).
Discussion
Fig. 7. Food intake in diabetic and non-diabetic rats, 8, 16 and 32 mg/kg ThT was administered to the treated groups. a: significantly different from control group (p < 0.05); b: significantly different from ThT2 (p < 0.05). Note: a and b refer to both diabetic and non-diabetic groups.
As mentioned before, new diabetic therapies seek ways to affect multiple targets at the same time with consideration of less side effects [4,7–9]. Our results indicate such a trend because administration of ThT in both diabetic and non-diabetic rats led to remarkable decrease in the level of blood glucose concentration and also meaningful reduction in the activity of alpha-amylase. Besides, administration of ThT at different doses resulted in the correction of lipid profile. Furthermore, ThT affected food intake and body weight too and these two parameters were decreased in
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Fig. 10. Superimposition of the two best docking poses of ThT in the active site of porcine pancreatic amylase. ThT is shown with balls and sticks, and the residues located in the 4.5 A˚ distance volume of the ligand are shown as lines.
all treated groups, which might be related to a potential appetite suppressing effect, as observed on another benzothiazole compound, as well as polyphenolic compounds [17,18,20]. The aforesaid findings show the complementarily therapeutic effect of ThT in diabetes. There are some probable hypotheses on the hypoglycemic mechanism of ThT. Our research paradigm was based on the partial inhibition of alpha-amylase by ThT. Carbohydratehydrolyzing enzymes’ inhibitors such as alpha-glucosidase inhibitors and alpha-amylase ones play an important role in decreasing postprandial hyperglycemia by prolonging the process of carbohydrate digestion [16,21,22]. The noteworthiness of this role can be highlighted by consideration of seriously detrimental impacts of postprandial hyperglycemia and especially cardiovascular damages [23,24]. Many studies were recently designed to find new inhibitors of alpha-amylase. Medicinal plants’ derivatives were within the scope of several studies including grape skin extract [25], a medicinal mushroom named Grifola frondosa [26], a kind of algae named Padina arboresens [22] and streptomycete endophytes from western Ghats [27]. Other studies have focused on small synthetic molecules that inhibit alpha-amylase such as glycoconjugated 1H-1,2,3-triazoles (GCTs) [28], glucopyranosylidene-spiro-thiohydantoin (G-TH) [29], sulfonium-ion glucosidase inhibitors [30] and also our recent study. As illustrated in Fig. 5, in both diabetic and non-diabetic groups, serum alphaamylase levels were decreased. The maximum inhibition was observed in DTh8, DTh16 and DTh32 in diabetics and in NDTh16 and NDTh32 in non-diabetic groups that showed non-dosedependent effect of ThT which may occur due to the saturation in the inhibitory property of ThT towards alpha-amylase similar to our previous experience on trans-chalcone [16]. However, administration of P. arboresens compounds and total flavonoids from Polygonatum odoratum in two other research studies in STZinduced diabetic mice indicated a clear dose-dependent pattern in the inhibition of alpha-amylase as well as greater inhibitory effect in comparison with our study [22,31]. The magnitude of maximum blood glucose reduction at the dose of 32 mg/kg of body weight in diabetic rats of our study, at around 38%, was greater than our previous experiences with the same therapeutic doses of trans-chalcone and citral by approximately 33 and 25% of
maximum reduction, respectively [16]. The percentage of maximum blood glucose reduction with ThT was almost the same as the other studies conducted with P. arboresens and Meliacinolin in diabetic rats [22,32]. Interestingly, ThT lessened the amount of blood glucose in non-diabetic rats too, regardless of blood glucose level. Moreover, we made a docking experiment in order to investigate the interaction between ThT and alpha-amylase. Relative to the docked pose that we have previously found for the alpha-amylase competitive inhibitor trans-chalcone [16,18] and the actual pose of myricetin, which is a flavonoid and inhibitor of alpha-amylase (4GQR.pdb), ThT was found to adopt a perpendicular orientation in alpha-amylase active site. The in vitro results suggest a definitive inhibitory effect of ThT on alpha-amylase, although it is possible that these compounds’ derivatives would have a better effect in this regard. There is another hypothesis that ThT may partially inhibit the formation of amyloids which could be responsible for the pathogenesis of type II diabetes. It means that ThT may play a therapeutic role beyond its familiar characteristic as a significant probe of amyloid formation [16,17]. A recent interesting study indicated that administration of ThT in Caenorhabditis elegans led to prolongation of life span by preserving protein homeostasis. The study proposed that this effect is due to the interaction between ThT and protein fibrils which causes slowing down and inhibition in the process of aggregation [33]. According to the findings of Xiangdong Su et al., benzothiazole derivatives are selective inhibitors of human 11-hydroxysteroid dehydrogenase type 1. In consequence, these derivatives inhibit conversion of cortisone to active glucocorticoid cortisol that has opposite action of insulin. Therefore, benzothiazole derivatives can be used as hypoglycemic agents [34]. And needless to say, ThT consists of a positively charged benzothiazole ring in its biochemical structure [35]. In conclusion, ThT shows remarkable therapeutic benefits, by taking into consideration that this small molecule simultaneously affects multiple anti-diabetic targets as shown by alpha-amylase partial inhibition, significant reduction in blood glucose, correction of lipid profile especially of triglyceride and cholesterol and also weight loss, further investigations on ThT and its derivatives may lead to the proposal of effective new anti-diabetics.
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Conflict of interests The authors declare that they have no competing interests. Acknowledgment The support from Endocrinology and Metabolism Research Institute of Tehran University of Medical Sciences is acknowledged. References [1] Forouzanfar MH, Sepanlou SG, Shahraz S, Dicker D, Naghavi P, Pourmalek F, et al. Evaluating causes of death and morbidity in Iran, global burden of diseases, injuries, and risk factors study 2010. Arch Iran Med 2014;17(5):304–20. [2] Skrha J. Diabetes mellitus-a global pandemic Keynote lecture presented at the Wonca conference in Prague in June 2013. Eur J Gen Pract 2014;20(1):65–8. [3] Whiting DR, Guariguata L, Weil C, Shaw J. IDF diabetes atlas: global estimates of the prevalence of diabetes for 2011 and 2030. Diabetes Res Clin Pract 2011;94(3):311–21. [4] Dey B, Mitra A, Katakam P, Singla RK. Exploration of natural enzyme inhibitors with hypoglycemic potentials amongst Eucalyptus Spp. by in vitro assays. World J Diabetes 2014;5(2):209–18. [5] Dong Y, Jing T, Meng Q, Liu C, Hu S, Ma Y, et al. Studies on the antidiabetic activities of cordyceps militaris extract in diet-streptozotocin-induced diabetic Sprague–Dawley rats. Biomed Res Int 2014;2014:160980. [6] Mitra A, Dewanjee D, Dey B. Mechanistic studies of lifestyle interventions in type 2 diabetes. World J Diabetes 2012;3(12):201–7. [7] DeFronzo RA. Pathogenesis of type 2 diabetes mellitus. Med Clin North Am 2004;88(4). 787–835, ix. [8] El-Abhar HS, Schaalan MF. Phytotherapy in diabetes: review on potential mechanistic perspectives. World J Diabetes 2014;5(2):176–97. [9] Lopez-Candales A. Metabolic syndrome X: a comprehensive review of the pathophysiology and recommended therapy. J Med 2001;32(5–6):283–300. [10] Kuznetsova IM, Sulatskaya AI, Uversky VN, Turoverov KK. A new trend in the experimental methodology for the analysis of the thioflavin T binding to amyloid fibrils. Mol Neurobiol 2012;45(3):488–98. [11] LeVine III H. Thioflavine T interaction with synthetic Alzheimer’s disease betaamyloid peptides: detection of amyloid aggregation in solution. Protein Sci 1993;2(3):404–10. [12] LeVine III H. Quantification of beta-sheet amyloid fibril structures with thioflavin T. Methods Enzymol 1999;309:274–84. [13] Vassar PS, Culling CF. Fluorescent stains, with special reference to amyloid and connective tissues. Arch Pathol 1959;68:487–98. [14] Ali H, Houghton PJ, Soumyanath A. Alpha-amylase inhibitory activity of some Malaysian plants used to treat diabetes: with particular reference to Phyllanthus amarus. J Ethnopharmacol 2006;107(3):449–55. [15] Machius M, Vertesy L, Huber R, Wiegand G. Carbohydrate and protein-based inhibitors of porcine pancreatic alpha-amylase: structure analysis and comparison of their binding characteristics. J Mol Biol 1996;260(3):409–21. [16] Najafian M, Ebrahim-Habibi A, Yaghmaei P, Parivar K, Larijani B. Core structure of flavonoids precursor as an antihyperglycemic and antihyperlipidemic agent: an in vivo study in rats. Acta Biochim Pol 2010;57(4):553–60.
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Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Thioflavin T effect in diabetic Wistar rats: Reporting the antihyperglycemic property of an amyloid probing dye Mahmood Najafian a, Shahab Amini b,1, Babak Dehestani c,1, Kazem Parivar d, Azadeh Ebrahim-Habibi e,c,* a
Department of Biology, Jahrom Branch, Islamic Azad University, Jahrom, Iran Diabetes Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran d Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran e Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran b c
A R T I C L E I N F O
Article history: Received 10 August 2014 Received in revised form 18 October 2014 Accepted 20 October 2014 Available online 5 November 2014 Keywords: Thioflavin T Diabetes Streptozotocin Amyloid Amylase.
A B S T R A C T
Background: Thioflavin T (ThT) is a well-known probe of amyloid fibrils with a benzothiazole core structure. As a compound with partial inhibitory effect on alpha-amylase, the results of oral ThT administration were investigated on a streptozotocin (STZ)-induced rat model of diabetes. Methods: STZ was administered intraperitoneally for induction of diabetes. Afterwards, doses of 2, 8, 16, and 32 mg/kg of ThT were used in diabetic and non-diabetic rats. Blood glucose levels, lipid profiles, alpha-amylase activity, food and water intake and urine volume were assessed. Docking was also performed to evaluate the inhibitory effect of ThT on alpha-amylase. Results: Upon treatment with ThT, blood glucose levels and lipid profile of diabetic rats improved significantly. Furthermore, alpha-amylase serum levels of treated animals decreased compared to the control group, suggesting a possible effect of ThT on this digestive enzyme. On the other hand, the food intake of treated animals showed a decrease. ThT effects were also seen to some extent in the nondiabetic group. Conclusion: ThT is suggested to be a potentially useful compound in treatment and prevention of diabetes and associated complications. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Diabetes is one of the leading causes of mortality and morbidity in the modern world and reported to be among top causes of death in the recent years [1,2]. According to the statistics of International Diabetes Federation (IDF), the predicted prevalence of diabetes suggests that approximately 592 million people will suffer from diabetes by 2030 [3]. Furthermore, as a chronic disease, diabetes is accompanied with serious kinds of complications such as vasculopathy, nephropathy, neuropathy and retinopathy. These realities show the vital importance of efficient therapeutic methods [4–6]. Conventional
* Corresponding author. E-mail addresses: [email protected], [email protected] (A. Ebrahim-Habibi). 1 These authors have contributed equally to the work.
hypoglycemic agents usually focus on one specific pathway to fight against diabetes, while as a multi-factorial illness it would be preferable that a drug affected multiple targets simultaneously [4,7,8]. Besides, diabetic patients have to tolerate life-long medication with important side effects such as hypoglycemia, hepatocellular injury, neurological deficit, diarrhea, nausea, vomiting, flatulence while they may finally suffer from lack of appropriate response to conventional hypoglycemic agents in the long term [5,8,9]. Therefore, there is a growing necessity to find new antidiabetic drugs with the aid of well-planned research studies [6,8,9]. In this study, we focused on the characteristics of a small molecule named Thioflavin T (ThT). ThT is a benzothiazole dye with IUPAC (International Union of Pure and Applied Chemistry nomenclature) name of 4-(3,6-dimethyl-1,3-benzothiazol-3-ium2-yl)-N,N-dimethylaniline chloride [10,11]. It has been widely used as a defining probe to detect amyloid fibrils, based on its distinctive property of displaying dramatically enhanced fluorescence in its interaction with amyloid fibril, since 1959 [10–13].
http://dx.doi.org/10.1016/j.pharep.2014.10.013 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
M. Najafian et al. / Pharmacological Reports 67 (2015) 364–369
Among latest strategies of controlling diabetes, inhibition of mammalian alpha-amylase is recently considered as a therapeutic plan for the disease by lessening postprandial hyperglycemia [14–17]. Our research paradigm was based upon the hypothesis that, with regard to its structure, ThT may be a possible human alphaamylase inhibitor. Thus, we did docking studies to investigate the interaction between alpha-amylase and ThT and also evaluated the remedial effects of ThT’s administration in rats as an in vivo model.
Materials and methods Chemicals THT, dimethyl sulfoxide, soluble starch and maltose were obtained from Merck (Darmstadt, Germany). Streptozotocin, porcine pancreatic alpha-amylase (PPA) (E.C.3.2.1.1) and dinitrosalycilic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Animals All rats were male Wistar, weighting 200 15 g and 2.5 months age. They were kept in cages in groups of six, inside a room at temperature of 22–24 8C. During daylight hours (8:00 to 20:00), room lights were turned on. Following standard rodent diet was used: maintenance diet Letica, Panlab S.L., Barcelona, Spain; 61.4% (w/w) carbohydrate (100% starch), 3.9% fiber, 15.1% protein and 2.7% fat, and tap water. Rats were given ad libitum access to food and water. Induction of diabetic condition was performed by means of a single-dose streptozotocin’s (STZ) administration (70 mg/kg body weight) dissolved in a citrate buffer (0.1 mol/l), pH 4.5. The administration of STZ was intraperitoneal and was performed according to existing protocols as used in previously published related work [16]. Afterwards, we measured blood glucose concentration every 2 days. Rats were divided into two major groups, ‘‘non-diabetic rats’’ (ND) and ‘‘diabetic rats’’ (D), and each was subsequently divided into five subgroups, as defined below: non-diabetic control group (NDC) was given ligand solvent, distilled water (O) orally during 24 days with the use of a gastric cannula in single doses of 0.5 ml at 8:30 AM. Diabetic control group (DC) was administered with the use of a gastric cannula in single doses of 0.5 ml at 8:30 AM, during 24 days. Non-diabetic group receiving ThT (NDTh): ThT was administered orally at 2, 8, 16 and 32 mg/kg (groups were named NDTh2, NDTh8, NDTh16 and NDTh32, respectively): the compound was dissolved in distilled water, and given during 24 days with the use of a gastric cannula in single doses of 0.5 ml at 8:30 AM. Diabetic groups receiving ThT (DTh): ThT was administered at 2, 8, 16 and 32 mg/kg body weight (respectively DTh2, DTh8, DTh16 and DTh32) dissolved in distilled water, orally, during 24 days through a gastric cannula in single doses of 0.5 ml at 8:30 AM. Since this project was, to our knowledge, the first animal experiment on anti-diabetic therapeutic effects of ThT, the doses were chosen based on our in vitro experiences with ThT, as well as considering our previous study on trans-chalcone whose in vitro and in vivo effects were both studied as a potential hypoglycemic agent [16,18]. The ethical aspect of the experimental protocol was given ethical clearance approval (code: E-0033) by the ethics committee of the Endocrinology and Metabolism Research Center as founder of the project, and the ethical clearance was also approved by the internal ethical committee of the Science and Research Branch of Islamic Azad University, Tehran, where the research was conducted.
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Measured parameters Food and water intake and urine volume were assessed and quantified every morning at 9:00 AM. Blood glucose levels were measured in blood samples that were obtained from the tail of the animal every 2 days on mornings at 9:00 AM with a glucometer (One Touch Profile, Life Scan). Body weight of the rats was measured after day 24, and the animals were then sacrificed under light ether anesthesia. Blood samples collected from rat hearts were placed on ice and centrifuged within 15 min after blood collection at 3000 g for 5 min. The concentrations of cholesterol, triacylglycerol, high-density lipoproteins (HDL), low-density lipoproteins (LDL) and alpha-amylase activity were measured with standard biochemical kits at the end of our experiment (day 24). Estimates of very-low-density lipoprotein (VLDL) were calculated from the formula VLDL-C = triacylglycerol/5 [13]. Statistical analysis The data were analyzed by means of one-sample Kolmogrov– Smirnov test and Levene’s test. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons were used to compare difference between experimental groups. The criterion for statistical significant was p < 0.05. Enzyme inhibition experiment The process of enzyme assay was conducted by use of the Bernfeld method, as previously described [18]. Soluble starch was the substrate and the activity of alpha-amylase was defined as one unit being the amount of enzyme able to release 1 mmol of maltose from starch per minute. Enzyme remaining activity in the presence of different concentrations of thioflavin was calculated as a percentage by comparison with the control sample activity. Docking Docking was performed with AutodockVina and use of the Gasteiger charges for the receptor and ligand [19]. Receptor was the 1DHK.pdb file which had been processed with the use of MOE 2010.10 (Chemical Computing Group Inc., Montreal, Canada), and the protonation state of the structure was adjusted for neutral pH. A Grid box of 40 40 40 points was used with a spacing 1.0 A˚, with the grid box center was put on x = 102.186, y = 40.132 and z = 18.012. Preparation of the image representing the best pose was done with MOE 2010.10.
Results Glucose During the whole experience, the consequence of induction of diabetes in diabetic control groups was a notable increase in the level of blood glucose (408.6 3.8) compared with non-diabetic group (107.9 4.1). Treatment with ThT resulted into a significant subside in the level of glucose concentrations in diabetic rats (p < 0.05). As shown in Fig. 1, in all groups, this decrease was steep until the 6th day of administration of thioflavin, followed by slight decrease in all doses by 332.2, 273.1, 250.3 and 237.6 mg/ml in DTh2 DTh8, DTh16 and DTh32 groups, respectively. A similar trend was seen in non-diabetic groups. However, there were no significant differences between NDTh8, NDTh16 and NDTh32 (Fig. 2).
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Fig. 1. Blood glucose levels in the diabetic group (D); DO was the control group and 2, 8, 16 and 32 mg/kg ThT was administered to the treated groups.
Lipids There was a significant increase in the level of all lipid profiles in diabetic control groups (50.9, 71.9, 70.6 and 80.5% in cholesterol, TG, VLDL and LDL, in order), while HDL decreased 39.2%. Administration of ThT in different doses resulted in decrease in all lipids’ concentrations (Figs. 3 and 4). Although there were no significant differences between DTh8, DTh16 and DTh32 groups, these three groups showed a dramatic (p < 0.05) decrease in cholesterol and triglyceride levels in comparison with the first two groups both in non-diabetic and diabetic groups. Furthermore, by administration of 16 mg/kg ThT, HDL concentration recovered to normal level of 41.6 after the experience (p < 0.05). In non-diabetic groups, there were no noteworthy changes in VLDL, LDL and HDL (p > 0.05). To sum up, the ideal dose of ThT administration for correcting lipid profile was found to be 8 mg/kg.
Fig. 3. Lipid profile of the diabetic group; 8, 16 and 32 mg/kg ThT was administered to the treated groups. a: significantly different from diabetic control group!cholesterol; b: significantly different from diabetic Th2 group!cholesterol; g: significantly different from diabetic control group!triglyceride.
Alpha-amylase As illustrated in Fig. 5, in both diabetic and non-diabetic groups, serum alpha-amylase levels were decreased. The maximum inhibition was observed in DTh8, DTh16 and DTh32 (p < 0.05) in diabetics and in NDTh8, NDTh16 and NDTh32 in non-diabetic groups (p < 0.05). Changes in DTh8, DTh16 and DTh32 groups were significantly different from DTh2 and control group (p < 0.05).
Fig. 2. Blood glucose levels in the non-diabetic group (ND); NDO was the control group and 2, 8, 16 and 32 mg/kg ThT was administered to the treated groups.
Fig. 4. Lipid profile of the non-diabetic group; 8, 16 and 32 mg/kg ThT was administered to the treated groups.
Fig. 5. Alpha-amylase serum levels in diabetic and non-diabetic groups, 8, 16 and 32 mg/kg ThT was administered to the treated groups. Exponential trend line is also indicated. a: significantly different from non-diabetic control group; b: significantly different from non-diabetic Th2; a (italic): significantly different from diabetic control group; b (italic): significantly different from diabetic Th2; g (italic): significantly different from Th16.
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Fig. 6. Body weights of diabetic and non-diabetic rats, 8, 16 and 32 mg/kg ThT was administered to the treated groups. a: significantly different from diabetic control group; b: significantly different from diabetic Th2 group.
Body weight and food intake
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Fig. 8. Urine volume in diabetic and non-diabetic rats, 8, 16 and 32 mg/kg ThT was administered to the treated groups. a: significantly different from diabetic control group; b: significantly different from diabetic Th2 group; g: significantly different from diabetic Th8 group.
Use of thioflavin probably deemed to have appetite-suppressing property in both diabetic and non-diabetic groups. Additionally, as can be seen in Figs. 6 and 7, weight as an associated factor with food intake was decreased significantly in all treated groups compared with control group (p < 0.05). However, the minimum food intake among diabetic groups was noticed in DTh8, DTh16 and DTh32 groups. Higher food consumption of diabetic groups compared with non-diabetics would be a characteristic of the disease state. Urine and water intake As a consequence of being in diabetic states, diabetic rats tend to drink more water and urinate more compared with the other group. Figs. 8 and 9 depict the amount of water consumed by rats. Diabetic control group consumed 110.5 ml/day which is approximately three times more than non-diabetic control group. After administration of ThT, significant decrease in both parameters was seen.
Fig. 9. Water intake in diabetic and non-diabetic rats, 8, 16 and 32 mg/kg ThT was administered to the treated groups.
Probing the interaction of ThT with alpha-amylase In silico docking experiment showed that thioflavin T is able to interact with the residues located in the binding site cleft of mammalian alpha-amylase. The docking results provided
56 poses whose energies ranged from 6.1 to 3.5 kcal/mol. The best two poses (with lowest energies) are shown in Fig. 10. The two positioning differ from each other with regard to the location of the benzothiazole ring, but show similar conformations. In the absence of an amylase crystal structure containing thioflavin T, both these orientations seem plausible. Upon testing ThT inhibitory activity toward a mammalian alphaamylase in vitro, primary results were the observation of a partial inhibitory effect, with a maximal inhibition of 80% with the use of a 400 micromolar concentration. Further experiments showed a lower inhibitory potential, which seemed to have a non dosedependent pattern (results not shown).
Discussion
Fig. 7. Food intake in diabetic and non-diabetic rats, 8, 16 and 32 mg/kg ThT was administered to the treated groups. a: significantly different from control group (p < 0.05); b: significantly different from ThT2 (p < 0.05). Note: a and b refer to both diabetic and non-diabetic groups.
As mentioned before, new diabetic therapies seek ways to affect multiple targets at the same time with consideration of less side effects [4,7–9]. Our results indicate such a trend because administration of ThT in both diabetic and non-diabetic rats led to remarkable decrease in the level of blood glucose concentration and also meaningful reduction in the activity of alpha-amylase. Besides, administration of ThT at different doses resulted in the correction of lipid profile. Furthermore, ThT affected food intake and body weight too and these two parameters were decreased in
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Fig. 10. Superimposition of the two best docking poses of ThT in the active site of porcine pancreatic amylase. ThT is shown with balls and sticks, and the residues located in the 4.5 A˚ distance volume of the ligand are shown as lines.
all treated groups, which might be related to a potential appetite suppressing effect, as observed on another benzothiazole compound, as well as polyphenolic compounds [17,18,20]. The aforesaid findings show the complementarily therapeutic effect of ThT in diabetes. There are some probable hypotheses on the hypoglycemic mechanism of ThT. Our research paradigm was based on the partial inhibition of alpha-amylase by ThT. Carbohydratehydrolyzing enzymes’ inhibitors such as alpha-glucosidase inhibitors and alpha-amylase ones play an important role in decreasing postprandial hyperglycemia by prolonging the process of carbohydrate digestion [16,21,22]. The noteworthiness of this role can be highlighted by consideration of seriously detrimental impacts of postprandial hyperglycemia and especially cardiovascular damages [23,24]. Many studies were recently designed to find new inhibitors of alpha-amylase. Medicinal plants’ derivatives were within the scope of several studies including grape skin extract [25], a medicinal mushroom named Grifola frondosa [26], a kind of algae named Padina arboresens [22] and streptomycete endophytes from western Ghats [27]. Other studies have focused on small synthetic molecules that inhibit alpha-amylase such as glycoconjugated 1H-1,2,3-triazoles (GCTs) [28], glucopyranosylidene-spiro-thiohydantoin (G-TH) [29], sulfonium-ion glucosidase inhibitors [30] and also our recent study. As illustrated in Fig. 5, in both diabetic and non-diabetic groups, serum alphaamylase levels were decreased. The maximum inhibition was observed in DTh8, DTh16 and DTh32 in diabetics and in NDTh16 and NDTh32 in non-diabetic groups that showed non-dosedependent effect of ThT which may occur due to the saturation in the inhibitory property of ThT towards alpha-amylase similar to our previous experience on trans-chalcone [16]. However, administration of P. arboresens compounds and total flavonoids from Polygonatum odoratum in two other research studies in STZinduced diabetic mice indicated a clear dose-dependent pattern in the inhibition of alpha-amylase as well as greater inhibitory effect in comparison with our study [22,31]. The magnitude of maximum blood glucose reduction at the dose of 32 mg/kg of body weight in diabetic rats of our study, at around 38%, was greater than our previous experiences with the same therapeutic doses of trans-chalcone and citral by approximately 33 and 25% of
maximum reduction, respectively [16]. The percentage of maximum blood glucose reduction with ThT was almost the same as the other studies conducted with P. arboresens and Meliacinolin in diabetic rats [22,32]. Interestingly, ThT lessened the amount of blood glucose in non-diabetic rats too, regardless of blood glucose level. Moreover, we made a docking experiment in order to investigate the interaction between ThT and alpha-amylase. Relative to the docked pose that we have previously found for the alpha-amylase competitive inhibitor trans-chalcone [16,18] and the actual pose of myricetin, which is a flavonoid and inhibitor of alpha-amylase (4GQR.pdb), ThT was found to adopt a perpendicular orientation in alpha-amylase active site. The in vitro results suggest a definitive inhibitory effect of ThT on alpha-amylase, although it is possible that these compounds’ derivatives would have a better effect in this regard. There is another hypothesis that ThT may partially inhibit the formation of amyloids which could be responsible for the pathogenesis of type II diabetes. It means that ThT may play a therapeutic role beyond its familiar characteristic as a significant probe of amyloid formation [16,17]. A recent interesting study indicated that administration of ThT in Caenorhabditis elegans led to prolongation of life span by preserving protein homeostasis. The study proposed that this effect is due to the interaction between ThT and protein fibrils which causes slowing down and inhibition in the process of aggregation [33]. According to the findings of Xiangdong Su et al., benzothiazole derivatives are selective inhibitors of human 11-hydroxysteroid dehydrogenase type 1. In consequence, these derivatives inhibit conversion of cortisone to active glucocorticoid cortisol that has opposite action of insulin. Therefore, benzothiazole derivatives can be used as hypoglycemic agents [34]. And needless to say, ThT consists of a positively charged benzothiazole ring in its biochemical structure [35]. In conclusion, ThT shows remarkable therapeutic benefits, by taking into consideration that this small molecule simultaneously affects multiple anti-diabetic targets as shown by alpha-amylase partial inhibition, significant reduction in blood glucose, correction of lipid profile especially of triglyceride and cholesterol and also weight loss, further investigations on ThT and its derivatives may lead to the proposal of effective new anti-diabetics.
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Conflict of interests The authors declare that they have no competing interests. Acknowledgment The support from Endocrinology and Metabolism Research Institute of Tehran University of Medical Sciences is acknowledged. References [1] Forouzanfar MH, Sepanlou SG, Shahraz S, Dicker D, Naghavi P, Pourmalek F, et al. Evaluating causes of death and morbidity in Iran, global burden of diseases, injuries, and risk factors study 2010. Arch Iran Med 2014;17(5):304–20. [2] Skrha J. Diabetes mellitus-a global pandemic Keynote lecture presented at the Wonca conference in Prague in June 2013. Eur J Gen Pract 2014;20(1):65–8. [3] Whiting DR, Guariguata L, Weil C, Shaw J. IDF diabetes atlas: global estimates of the prevalence of diabetes for 2011 and 2030. Diabetes Res Clin Pract 2011;94(3):311–21. [4] Dey B, Mitra A, Katakam P, Singla RK. Exploration of natural enzyme inhibitors with hypoglycemic potentials amongst Eucalyptus Spp. by in vitro assays. World J Diabetes 2014;5(2):209–18. [5] Dong Y, Jing T, Meng Q, Liu C, Hu S, Ma Y, et al. Studies on the antidiabetic activities of cordyceps militaris extract in diet-streptozotocin-induced diabetic Sprague–Dawley rats. Biomed Res Int 2014;2014:160980. [6] Mitra A, Dewanjee D, Dey B. Mechanistic studies of lifestyle interventions in type 2 diabetes. World J Diabetes 2012;3(12):201–7. [7] DeFronzo RA. Pathogenesis of type 2 diabetes mellitus. Med Clin North Am 2004;88(4). 787–835, ix. [8] El-Abhar HS, Schaalan MF. Phytotherapy in diabetes: review on potential mechanistic perspectives. World J Diabetes 2014;5(2):176–97. [9] Lopez-Candales A. Metabolic syndrome X: a comprehensive review of the pathophysiology and recommended therapy. J Med 2001;32(5–6):283–300. [10] Kuznetsova IM, Sulatskaya AI, Uversky VN, Turoverov KK. A new trend in the experimental methodology for the analysis of the thioflavin T binding to amyloid fibrils. Mol Neurobiol 2012;45(3):488–98. [11] LeVine III H. Thioflavine T interaction with synthetic Alzheimer’s disease betaamyloid peptides: detection of amyloid aggregation in solution. Protein Sci 1993;2(3):404–10. [12] LeVine III H. Quantification of beta-sheet amyloid fibril structures with thioflavin T. Methods Enzymol 1999;309:274–84. [13] Vassar PS, Culling CF. Fluorescent stains, with special reference to amyloid and connective tissues. Arch Pathol 1959;68:487–98. [14] Ali H, Houghton PJ, Soumyanath A. Alpha-amylase inhibitory activity of some Malaysian plants used to treat diabetes: with particular reference to Phyllanthus amarus. J Ethnopharmacol 2006;107(3):449–55. [15] Machius M, Vertesy L, Huber R, Wiegand G. Carbohydrate and protein-based inhibitors of porcine pancreatic alpha-amylase: structure analysis and comparison of their binding characteristics. J Mol Biol 1996;260(3):409–21. [16] Najafian M, Ebrahim-Habibi A, Yaghmaei P, Parivar K, Larijani B. Core structure of flavonoids precursor as an antihyperglycemic and antihyperlipidemic agent: an in vivo study in rats. Acta Biochim Pol 2010;57(4):553–60.
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