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research-article2014
DVR0010.1177/1479164114521643Diabetes & Vascular Disease ResearchOliveira et al.
Original Article
Phaseolamin treatment prevents oxidative stress and collagen deposition in the hearts of streptozotocin-induced diabetic rats
Diabetes & Vascular Disease Research 2014, Vol. 11(2) 110–117 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1479164114521643 dvr.sagepub.com
Renato JS Oliveira1, Vanessa N de Oliveira1,2, Simone R Deconte1, Luciana K Calábria1,3, Alberto da Silva Moraes4, and Foued S Espindola1
Abstract The development of cardiovascular complications in patients with diabetes is often associated with an imbalance between reactive oxygen species and antioxidant systems. This imbalance can contribute to high cardiac collagen content, which increases cross-linking and the stiffness of the myocardium. In this study, the protective effect of phaseolamin against damage under oxidative stress and collagen deposition in the cardiac tissue in association with diabetes was evaluated. Non-diabetic and diabetic animals were distributed into groups and treated for 20 days with commercial phaseolamin. The phaseolamin treatment increased total antioxidant activity but reduced the following in diabetic rats: (a) hyperglycaemic state, (b) catalase and superoxide dismutase activity and (c) tissue damage caused by lipid peroxidation. Additionally, the phaseolamin treatment attenuated the collagen levels compared to non-treated diabetic rats. Thus, the short-term anti-hyperglycaemic effect of the phaseolamin treatment may prevent the initial changes caused by oxidative stress and the deposition of collagen, as well as reduce the incidence of heart complications. Keywords Diabetes, oxidative stress, alpha-amylase inhibitor, collagen, phaseolamin
Introduction Diabetes is a major risk factor for the development of cardiovascular complications that now accounts for 80% of all mortality.1 Evidence from experimental models shows that the elevated extra- and intracellular glucose levels observed with diabetes increase the imbalance between pro-oxidants and antioxidants, i.e. oxidative stress is responsible for complications such as cardiomyopathy.2–4 Such studies also show that the formation of advanced glycation end products (AGEs) on extracellular matrix components leads to an increase in collagen cross-linking, which contributes to myocardial stiffness that occurs concomitantly with diabetes.5–7 Thus, an increase in antioxidant enzymes in the hyperglycaemic state may be a consequence of the increased production of reactive oxygen species (ROS) in some tissues and organs, including the heart.8 The drugs classically used for treating diabetes are insulin, sulfonylureas, biguanides and thiazolidinediones. Alternative treatments, including antioxidant supplements, are used to control diabetic complications and have been reported to minimize the occurrence of cardiac
complications and reduce the functional and morphological damage to the diabetic heart.2 Phaseolus vulgaris has a constituent protein called phaseolamin that has an inhibitory effect on amylase. Phaseolamin is the focus of many studies due to its capacity as an anti-hyperglycaemiant.9 One previous study showed that purified phaseolamin non-competitively inhibits the enzyme alpha-amylase.10 The inhibitory mechanism of 1Institute
of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia, Brazil 2Department of Anatomy, Institute of Science Biology, Federal University of Juiz de Fora, Juiz de Fora, Brazil 3Basic Department – Health Area, Federal University of Juiz de Fora, Governador Valadares, Brazil 4Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia, Brazil Corresponding author: Foued S Espindola, Laboratory of Biochemistry and Molecular Biology, Institute of Genetics and Biochemistry, Federal University of Uberlândia, Avenida Para 1720, Uberlândia, MG, 38400-902, Brazil. Email: [email protected]
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Oliveira et al. phaseolamin is similar to that of other alpha-amylase inhibitors due to the high similarity of amino acid sequences.10 This inhibitory effect of phaseolamin reduces the metabolism of starch and the incidence of hyperglycaemia in diabetes. Phaseolamin is available on the market at varying degrees of specific activity (depending on the commercial source) and is present in various diet supplements and dietetic products.11 Although the clinical features of diabetic heart disease have been identified, its pathogenesis has not been fully elucidated, particularly the mechanisms underlying the collagen abnormalities in the diabetic heart. Collagen type I represents the key phenotype expressed in the heart.12 The increase in interstitial collagen causes cardiac fibrosis and leads to increased stiffness of the left ventricle, which causes diastolic dysfunction and heart failure.13 The aim of this study was to evaluate the effect of phaseolamin on alterations of oxidative parameters and the collagen deposition in the heart of streptozotocin-induced diabetic rats.
phaseolamin (Nanjing Well Chemical Corp., Ltd, China) (D100, D500 and D1500, respectively). Treatments were administered once per day by oral gavage for 20 days. After the final treatment, all rats were anesthetized by an intraperitoneal injection of xylazine (10 mg/kg bw) and ketamine (75 mg/kg bw) and were then sacrificed by euthanasia to allow for tissue dissection.
Sample collection
Male Wistar rats (weight: 160–210 g) were housed under standard conditions (25°C ± 2°C, humidity 60% ± 5%, 12/12-h light/dark cycle) for a period of 2 weeks. Animals received standard extruded chow and water ad libitum until either treatment or sacrifice. All experimental procedures followed the guidelines proposed by the Brazilian Society of Laboratory Animal Science and were approved by the Ethics Committee for Animal Research of the Federal University of Uberlândia, Brazil (CEUA/UFU 051/08).
The hearts were quickly removed, washed with chilled 0.9% NaCl, weighed and either immersed in liquid nitrogen or fixed in 10% formaldehyde solution in 0.1 M phosphate-buffered saline (pH 7.4). The ratio of heart weight (mg) to body weight (g) was calculated. Blood was collected from the portal vein to measure blood glucose and serum biochemical parameters such as total cholesterol (using the enzymatic Trinder method), plasma triglycerides (using the enzymatic Trinder method), total protein (using the Biuret method), creatinine (using a modified Heinegard and Tiderstram’s method), urea [using the urease ultraviolet (UV) kinetic method], aspartate aminotransferase (AST) [using the International Federation of Clinical Chemistry (IFCC) UV kinetic method], alanine aminotransferase (ALT) (using the IFCC UV kinetic method), γ-glutamyltransferase (γ-GT) (using a modified Szasz method) and alkaline phosphatase (using a modified Bowers and McComb method). All parameters were measured in a clinical analysis laboratory at the Faculty of Veterinary Medicine of the Federal University of Uberlândia with a Cobas Mira automatic analyser (Roche Diagnostic Systems, Basel, Switzerland) at 37°C using commercially available kits (Labtest Diagnostica©, Lagoa Santa, MG, Brazil).
Diabetes induction
Homogenate preparation
Rats were starved for 24 h and were then anesthetized by an intraperitoneal injection of xylazine [10 mg/kg body weight (bw)] and ketamine (75 mg/kg bw). Diabetes was induced by injection of streptozotocin (single dose, 40 mg/ kg bw) in 0.01 M citrate buffer (pH 4.5) (Sigma–Aldrich, St. Louis, MO, USA) via the penile vein (2 mL/kg bw); 10 days after diabetes induction, the fasting blood glucose level was monitored using reactive strips (Biocheck Glucose Test Strip; Bioeasy, Belo Horizonte, MG, Brazil). Rats with a blood glucose level above 200 mg/dL were considered to be diabetic.
Each heart was homogenized separately on ice in homogenization buffer containing 40 mM HEPES, 100 mM ethylenediaminetetraacetic (EDTA), 2 mM ethylene glycol tetraacetic acid (EGTA), 2 mM dithiothreitol (DTT), 1 mM benzamidine and 0.5 mM phenylmethanesulphonylfluoride (PMSF). The homogenates were centrifuged at 10,000g for 10 min at 4°C, and the total protein concentration in the supernatants was measured by the Bradford14 assay. Supernatants were assayed for the activity of oxidative stress markers.
Methods Animals
Analysis of oxidative stress markers
Groups and treatments Rats were randomly divided into six groups (n = 8 rats/ group): non-diabetic (ND); non-treated diabetic (NTD); diabetic treated with 25 mg/kg acarbose (DACA) and diabetic treated with 100, 500 or 1500 mg/kg commercial
The levels of lipid peroxidation in the tissue were determined by measuring malondialdehyde (MDA) by the thiobarbituric acid reactive substances (TBARS) assay using a commercial kit (Cayman Chemical Company, Ann Arbor, MI, USA). The total antioxidant status (TAS) and
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ND: non-diabetic; NTD: non-treated diabetic; D100, D500 and D1500: diabetic rats treated with phaseolamin at 100, 500 and 1500 mg/kg, respectively; DACA: diabetic rats treated with acarbose; γ-GT: γ-glutamyltransferase; AST: aspartate aminotransferase; ALT: alanine aminotransferase. ap < 0.05 versus ND. bp < 0.001 versus ND. cp < 0.05 versus NTD.
284.00 ± 17.59 1047.50 ± 103.30 3.66 ± 0.13 397.66 ± 39.33b 82.68 ± 4.91 0.68 ± 0.07 359.08 ± 82.25a 17.21 ± 10.73 92.00 ± 6.76 78.00 ± 10.94a 117.13 ± 55.54a 61.13 ± 6.11a 301.00 ± 12.83 967.50 ± 35.67a 3.22 ± 0.13 333.57 ± 43.88b,c 71.41 ± 3.46 0.57 ± 0.09 261.65 ± 55.52c 6.20 ± 2.85 100.57 ± 8.94 62.28 ± 6.25 99.72 ± 17.66 48.88 ± 6.21c 270.75 ± 16.54 930.00 ± 25.49a 3.47 ± 0.25 415.40 ± 53.48b,c 65.60 ± 5.91 0.68 ± 0.66 319.18 ± 77.68 9.08 ± 3.42 120.80 ± 22.96 64.40 ± 15.66 125.40 ± 13.42 54.54 ± 4.41c Body weight (g) Heart weight (mg) Heart/body weight Glycaemia (mg/dL) Cholesterol (mg/dL) Creatinine (mg/dL) Alkaline phosphatase (U/L) γ-GT (U/L) AST (U/L) ALT (U/L) Triglycerides Urea (mg/dL)
319.75 ± 6.90 1305.00 ± 130.41 4.10 ± 0.46 104.12 ± 5.32 65.33 ± 4.99 0.62 ± 0.03 92.41 ± 7.45 5.38 ± 1.42 74.62 ± 3.52 36.37 ± 2.30 59.96 ± 9.57 37.26 ± 1.68
255.00 ± 16.29a 987.50 ± 20.15 3.97 ± 0.30 568.57 ± 38.04a 70.45 ± 4.72 0.87 ± 0.05 559.32 ± 62.14b 19.80 ± 9.35 116.71 ± 11.38 97.71 ± 6.19b 98.93 ± 40.60 77.68 ± 5.59b
233.00 ± 12.34a 877.50 ± 47.67b 3.79 ± 0.26 330.00 ± 85.68b,c 65.90 ± 2.09 0.66 ± 0.07 318.16 ± 19.77 10.11 ± 5.86 93.33 ± 10.84 76.50 ± 10.59a 114.53 ± 18.45 57.03 ± 5.22
DACA D500
D1500
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D100 NTD ND
Table 1. Body and heart weight, and biochemical parameters.
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superoxide dismutase (SOD) activity were measured using a commercial kit (Randox Laboratories Ltd, Crumlin, UK). Catalase (CAT) activity was assayed spectrophotometrically by monitoring hydrogen peroxide decomposition at 240 nm,15 with a substrate concentration of 20 mM for cardiac tissue measurements. All analyses were performed in duplicate, and samples were normalized to protein concentration.
Histology and measurement of collagen Heart tissue was fixed with 10% formaldehyde in 0.1 M phosphate-buffered saline (pH 7.4) for 24 h, dehydrated in ethanol, cleared in xylene and embedded in paraffin. To determine the percentage of connective tissue, 5-µm sections were stained with picrosirius red.16,17 Picrosirius red technique is used to analyse collagen content because it binds to collagen molecules other than type I, such as type III.18 Morphometric analysis was carried out using a light microscope (Olympus Biotech UK Ltd, Hertfordshire, UK) with a 40× objective, and the microscope was equipped with an Oly-200 charge-coupled device (CCD) camera linked to a personal computer (PC) with a captureand-image-analysis system (HL-Image 97; Western Vision Software, Layton, UT, USA). In total, 30 random fields within areas that had a higher proportion of staining were selected, and the percentage of collagen pixels per field area was measured.
Statistical analysis All the values are expressed as the mean ± standard error of mean (SEM). Data were analysed by one-way analysis of variance (ANOVA) using SigmaStat 3.5 software (Systat Software, Inc., Chicago, IL, USA); p < 0.05 was considered to be statistically significant.
Results Table 1 summarizes the mean changes in body and cardiac weight, as well as biochemical parameters such as glycaemia, cholesterol, creatinine, alkaline phosphatase, γ-GT, AST, ALT, triglycerides and urea. After 20 days, the NTD group presented lower body weight and increased glycaemia, alkaline phosphatase, ALT and urea levels compared to the ND group. Treatment with phaseolamin decreased the blood glucose of diabetic animals by 26% and 42% (D100 and D500 groups, respectively), and administration of acarbose caused a 30% glycaemic reduction. The serum activity of alkaline phosphatase was fivefold higher in the NTD group compared to the ND group. Groups of diabetic animals treated with phaseolamin showed a 44% and 54% reduction in enzyme activity (D100 and D1500 groups, respectively), and the DACA group showed a 36% reduction in the enzyme activity compared to the NTD group.
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Figure 1. The effect of diabetes and phaseolamin treatment on oxidative stress biomarker levels in rat hearts. (a) Total antioxidant activity: ‘a’ represents p < 0.001 versus other groups; ‘b’ represents p < 0.001 versus other groups. (b) Superoxide dismutase activity: ‘a’ represents p < 0.001 versus ND, D100, D500 and DACA. (c) Malondialdehyde concentration: ‘a’ represents p < 0.001 versus NTD, D100, D500 and DACA; ‘b’ represents p < 0.001 versus NTD, D100 and DACA. (d) Catalase activity: ‘a’ represents p < 0.001 versus other groups.
ND: non-diabetic; NTD; non-treated diabetic; D100, D500 and D1500: diabetic animals supplemented with phaseolamin at 100, 500 and 1500 mg/kg, respectively; DACA: diabetic treated with 25 mg/kg acarbose; MDA: malondialdehyde; SEM: standard error of mean. Bars represent the mean ± SEM.
The urea level was twofold higher in the NTD group compared to the ND group. The D500 and D1500 groups that were treated with phaseolamin had a reduced serum concentration of urea compared to the NTD group. No changes were observed in the cholesterol, creatine and AST levels. Enzymes involved in the antioxidant defence system were impaired in the hyperglycaemic state, resulting in a reduction of the total antioxidant activity of the NTD group compared to the ND group. However, treatment with acarbose and phaseolamin caused an increase in the TAS levels in cardiac tissue compared to the NTD group (Figure 1(a)). In contrast to the lack of an effect on the enzymatic defence system, TAS was negatively correlated to MDA concentration (r = −0.7485), CAT activity (r = −0.6545), sulfhydryl group linked to protein (r = −0.6371) and collagen content (r = −0.6322).
Under hyperglycaemic conditions, SOD activity was higher in the NTD group compared to the ND group (Figure 1(b)). Groups treated with either acarbose or phaseolamin (D100, D500 and DACA) had decreased SOD activity compared to the NTD group. We observed an increase in CAT activity in the NTD group compared to the ND group and the other diabetic groups that were treated (Figure 1(d)). There was no difference between diabetic animals that were treated (D100, D500, D1500 and DACA) compared to the ND group. However, correlation analysis showed that hyperglycaemia is positively correlated to CAT activity (r = 0.67). After induction of diabetes, the marker of lipid peroxidation products (MDA) was increased in the NTD, D100, D500 and DACA groups compared to the ND group. However, the MDA concentration of the D1500 group was similar to that of the ND group (Figure 1(c)).
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Figure 2. The deposition of collagen fibres in rat hearts. (a) Total content of collagen: *p < 0.001 versus NTD; **p < 0.05 versus NTD. (b) Histological sections of rat heart stained with picrosirius red.
ND: non-diabetic; NTD; non-treated diabetic; D100, D500 and D1500: diabetic animals supplemented with phaseolamin at 100, 500 and 1500 mg/kg, respectively; DACA: diabetic treated with 25 mg/kg acarbose; SEM: standard error of mean. Bars represent the mean ± SEM.
The total content of collagen was examined using the picrosirius red method (Figure 2). We observed that there was increased collagen deposition in the rat hearts from the NTD group. The D100 and D500 groups did not display any decrease in collagen content and had higher collagen concentrations than the ND group and similar concentrations to the NTD group. The D1500 and DACA groups showed reduced values of collagen compared to the NTD group, and no difference was observed compared to the ND group (Figure 2(a)). Groups treated with phaseolamin (D500, D1500 and DACA) displayed attenuation of the weight loss caused by diabetes, and these groups showed a strong negative correlation between weight and collagen deposition in the cardiac tissue (r = −0.80).
Discussion The increased oxidative stress in diabetes due to the overproduction of ROS may be related to an increase in collagen and the development of cardiovascular diseases such as diastolic stiffness.19,20 Phaseolamin, an inhibitor of the alpha-amylase enzyme extracted from white beans, has become more popular in the weight management industry and has been linked to a reduced risk of diabetes, obesity,21 coronary heart disease,22 colon cancer23 and gastrointestinal disorders.24 Our results revealed a marked weight decrease in diabetic animals, but no changes in the heart/body weight ratio were observed in these animals. The blood glucose of diabetic animals reached high levels; however, phaseolamin treatment was able to reduce glycaemia after 20 days. Phaseolamin has a mechanism similar to acarbose, which is based on delayed and/or prevented digestion of carbohydrates due to competitive inhibition of glucosidases.25 Furthermore, significant weight loss was observed after 20
days of streptozotocin induction with the exception of those treated with phaseolamin. Other studies have verified a reduction of weight at 3,26 2127 and 60 days28 after streptozotocin induction. The liver enzymes ALT and AST reveal possible liver damage. ALT is also a more specific indicator of liver inflammation and contributes to the cleavage of protein cross-linkage with collagen, thus reducing the stiffness of arteries and the heart.29 Low serum ALT levels were observed in rats treated with phaseolamin. It is possible that this treatment attenuates the hyperglycaemic condition and stiffness in the heart caused by cross-linking between collagen. Creatinine and urea were used as markers for renal injuries. Creatinine remained stable in both treated and nontreated rats, indicating preserved renal function. However, elevated urea levels were observed in diabetic rats, which may indicate initial damage caused by the hyperglycaemic state. Relative to oxidative stress, phaseolamin treatment increased total antioxidant activity and promoted a compensatory mechanism that reduced antioxidant enzyme function. Moreover, this treatment was able to attenuate lipid peroxidation in diabetic rats. CAT activity has been previously found to be elevated in several tissues and organs of diabetic rats, including the heart,30–33 aorta,30,34 liver,35 parotid glands,36 kidney and brain.37 This alteration of CAT activity is due to diabetes and may be normalized by treatments such as captopril, aminoguanidine,38 acetylsalicylic acid35 and dehydroepiandrosterone.37 However, 20 days of either phaseolamin or acarbose treatment was sufficient to reduce the cardiac CAT activity. It is possible that this downregulation generates an anti-hyperglycaemic effect that minimizes the ROS levels in the hearts of the rats. However, the effects of CAT are still somewhat
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Oliveira et al. conflicting because a recent study showed a protective role of overexpressed CAT that prevented the development of diseases such as myocyte hypertrophy, apoptosis and interstitial fibrosis.39 Distinct SOD activity had been observed in cardiac tissue: decreased SOD activity at 4 and 8 weeks after induction of diabetes40 and increased SOD activity at 32 weeks.41 Despite the fact that diabetes leads to increased SOD levels due to the increased dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide as an adaptive response to oxidative stress, phaseolamin and acarbose treatments reduce its activity. Streptozotocin-induced diabetes regularly results in an increase in MDA,30,42,43 which indicates indirect damage caused by free radicals and is associated with the onset of myocardial fibrosis.44 Phaseolamin treatment decreases cardiac MDA concentrations to levels similar to those of NDs. This antioxidant activity may be associated with phenolic compounds present in beans, either in isolation or in combination with other components that have antioxidant action.45 Histopathological examination showed a marked and significant increase in collagen deposition in diabetic rats as well as a reduction in this deposition in diabetic rats that received phaseolamin treatment, confirming its cardioprotective action. Recent reports have demonstrated the protective action of acarbose, showing beneficial effects on endothelial function,46 anti-hyperglycaemic action47 and cardioprotective effects.48 The hyperglycaemic state is the primary driver of the formation of AGEs49 and has a key role in the development of diabetes-related complications5 that affect the structure and function of the myocardium. These complications are generally attributed to increased collagen deposition in this tissue.50 Studies have shown that glucose plays an important role in the accumulation of collagen in tissues through the increase in collagen synthesis at the transcriptional level.51,52 The glycation process of collagen inhibits its degradation by interfering with the action of metalloproteinases53 and protein kinases activated by mitogen to further myocardial interstitial fibrosis.49,54 AGE formation modifies proteins by forming links between the amino groups and other proteins, which alters the structure of the extracellular matrix and leads to myocardial stiffness.53 CAT overexpression preserves cardiac morphology, prevents changes in contractile function and reduces the concentrations of MDA and AGEs.55 We showed increased activity of antioxidant enzymes (SOD and CAT) as well as lipid peroxidation in diabetic rats, but the increased activity of these enzymes alone was insufficient to reduce oxidative stress and collagen deposition in the heart. However, phaseolamin treatment attenuated the action of ROS and reduced the collagen content in the heart. This effect may be a result of decrease in AGEs’ concentration because glycation causes increased synthesis of various types of collagens.56
In summary, the short-term anti-hyperglycaemic effect of phaseolamin treatment may prevent initial changes caused by oxidative stress and the deposition of collagen as well as reduce the occurrence of heart complications. However, additional studies should be carried out in detail to evaluate any long-term clinical changes. Acknowledgements The authors thank the School of Veterinary Medicine, Federal University of Uberlândia; Prof. Dr Antonio Vicente Mundim and Felipe Cesar Gonçalves for their help in processing the biochemical analyses. The authors also thank Neire Moura de Gouveia, Fernanda Vieira Alves and Fabiana Barcelos Furtado for the technical support.
Declaration of conflicting interests The authors declare that there are no conflicts of interest.
Funding This study was funded by the Brazilian Governmental Agencies from Ministry of Health (Research Program for SUS, PPSUS) and FAPEMIG (#EDT-3257/06). RJSO, SRD and LKC received fellowship from CAPES, and FSE is a CNPq fellow.
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Oliveira et al. 43. Jung CH, Zhou S, Ding GX, et al. Antihyperglycemic activity of herb extracts on streptozotocin-induced diabetic rats. Biosci Biotechnol Biochem 2006; 70: 2556–2559. 44. Stehr CB, Mellado R, Ocaranza MP, et al. Increased levels of oxidative stress, subclinical inflammation, and myocardial fibrosis markers in primary aldosteronism patients. J Hypertens 2010; 28: 2120–2126. 45. Cardador-Martinez A, Loarca-Pina G and Oomah BD. Antioxidant activity in common beans (Phaseolus vulgaris L.). J Agric Food Chem 2002; 50: 6975–6980. 46. Asakura M, Kim J, Asanuma H, et al. Anti-diabetic drugs for secondary prevention of cardiovascular disease in mild diabetic and IGT patients: ABC study and PPAR study. Nihon Rinsho 2010; 68: 887–891. 47. Halimi S. Towards new recommendations for the treatment of type 2 diabetes? An expert’s point of view. Rev Prat 2010; 60: 504–509. 48. Vanuzzo D, Pilotto L, Mirolo R, et al. Cardiovascular risk and cardiometabolic risk: an epidemiological evaluation. G Ital Cardiol (Rome) 2008; 9: 6S–17S. 49. Liu J, Masurekar MR, Vatner DE, et al. Glycation endproduct cross-link breaker reduces collagen and improves cardiac function in aging diabetic heart. Am J Physiol Heart Circ Physiol 2003; 285: H2587–H2591.
50. Avendano GF, Agarwal RK, Bashey RI, et al. Effects of glucose intolerance on myocardial function and collagenlinked glycation. Diabetes 1999; 48: 1443–1447. 51. Turk Z, Misur I, Turk N, et al. Rat tissue collagen modified by advanced glycation: correlation with duration of diabetes and glycemic control. Clin Chem Lab Med 1999; 37: 813–820. 52. Reiser KM. Nonenzymatic glycation of collagen in aging and diabetes. Proc Soc Exp Biol Med 1991; 196: 17–29. 53. Kuzuya M, Asai T, Kanda S, et al. Glycation cross-links inhibit matrix metalloproteinase-2 activation in vascular smooth muscle cells cultured on collagen lattice. Diabetologia 2001; 44: 433–436. 54. Lakshmanan AP, Harima M, Sukumaran V, et al. Modulation of AT-1R/AMPK-MAPK cascade plays crucial role for the pathogenesis of diabetic cardiomyopathy in transgenic type 2 diabetic (Spontaneous Diabetic Torii) rats. Biochem Pharmacol 2012; 83: 653–660. 55. Ye G, Metreveli NS, Donthi RV, et al. Catalase protects cardiomyocyte function in models of type 1 and type 2 diabetes. Diabetes 2004; 53: 1336–1343. 56. Goldin A, Beckman JA, Schmidt AM, et al. Advanced glycation end products: sparking the development of diabetic vascular injury. Circulation 2006; 114: 597–605.
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research-article2014
DVR0010.1177/1479164114521643Diabetes & Vascular Disease ResearchOliveira et al.
Original Article
Phaseolamin treatment prevents oxidative stress and collagen deposition in the hearts of streptozotocin-induced diabetic rats
Diabetes & Vascular Disease Research 2014, Vol. 11(2) 110–117 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1479164114521643 dvr.sagepub.com
Renato JS Oliveira1, Vanessa N de Oliveira1,2, Simone R Deconte1, Luciana K Calábria1,3, Alberto da Silva Moraes4, and Foued S Espindola1
Abstract The development of cardiovascular complications in patients with diabetes is often associated with an imbalance between reactive oxygen species and antioxidant systems. This imbalance can contribute to high cardiac collagen content, which increases cross-linking and the stiffness of the myocardium. In this study, the protective effect of phaseolamin against damage under oxidative stress and collagen deposition in the cardiac tissue in association with diabetes was evaluated. Non-diabetic and diabetic animals were distributed into groups and treated for 20 days with commercial phaseolamin. The phaseolamin treatment increased total antioxidant activity but reduced the following in diabetic rats: (a) hyperglycaemic state, (b) catalase and superoxide dismutase activity and (c) tissue damage caused by lipid peroxidation. Additionally, the phaseolamin treatment attenuated the collagen levels compared to non-treated diabetic rats. Thus, the short-term anti-hyperglycaemic effect of the phaseolamin treatment may prevent the initial changes caused by oxidative stress and the deposition of collagen, as well as reduce the incidence of heart complications. Keywords Diabetes, oxidative stress, alpha-amylase inhibitor, collagen, phaseolamin
Introduction Diabetes is a major risk factor for the development of cardiovascular complications that now accounts for 80% of all mortality.1 Evidence from experimental models shows that the elevated extra- and intracellular glucose levels observed with diabetes increase the imbalance between pro-oxidants and antioxidants, i.e. oxidative stress is responsible for complications such as cardiomyopathy.2–4 Such studies also show that the formation of advanced glycation end products (AGEs) on extracellular matrix components leads to an increase in collagen cross-linking, which contributes to myocardial stiffness that occurs concomitantly with diabetes.5–7 Thus, an increase in antioxidant enzymes in the hyperglycaemic state may be a consequence of the increased production of reactive oxygen species (ROS) in some tissues and organs, including the heart.8 The drugs classically used for treating diabetes are insulin, sulfonylureas, biguanides and thiazolidinediones. Alternative treatments, including antioxidant supplements, are used to control diabetic complications and have been reported to minimize the occurrence of cardiac
complications and reduce the functional and morphological damage to the diabetic heart.2 Phaseolus vulgaris has a constituent protein called phaseolamin that has an inhibitory effect on amylase. Phaseolamin is the focus of many studies due to its capacity as an anti-hyperglycaemiant.9 One previous study showed that purified phaseolamin non-competitively inhibits the enzyme alpha-amylase.10 The inhibitory mechanism of 1Institute
of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia, Brazil 2Department of Anatomy, Institute of Science Biology, Federal University of Juiz de Fora, Juiz de Fora, Brazil 3Basic Department – Health Area, Federal University of Juiz de Fora, Governador Valadares, Brazil 4Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia, Brazil Corresponding author: Foued S Espindola, Laboratory of Biochemistry and Molecular Biology, Institute of Genetics and Biochemistry, Federal University of Uberlândia, Avenida Para 1720, Uberlândia, MG, 38400-902, Brazil. Email: [email protected]
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Oliveira et al. phaseolamin is similar to that of other alpha-amylase inhibitors due to the high similarity of amino acid sequences.10 This inhibitory effect of phaseolamin reduces the metabolism of starch and the incidence of hyperglycaemia in diabetes. Phaseolamin is available on the market at varying degrees of specific activity (depending on the commercial source) and is present in various diet supplements and dietetic products.11 Although the clinical features of diabetic heart disease have been identified, its pathogenesis has not been fully elucidated, particularly the mechanisms underlying the collagen abnormalities in the diabetic heart. Collagen type I represents the key phenotype expressed in the heart.12 The increase in interstitial collagen causes cardiac fibrosis and leads to increased stiffness of the left ventricle, which causes diastolic dysfunction and heart failure.13 The aim of this study was to evaluate the effect of phaseolamin on alterations of oxidative parameters and the collagen deposition in the heart of streptozotocin-induced diabetic rats.
phaseolamin (Nanjing Well Chemical Corp., Ltd, China) (D100, D500 and D1500, respectively). Treatments were administered once per day by oral gavage for 20 days. After the final treatment, all rats were anesthetized by an intraperitoneal injection of xylazine (10 mg/kg bw) and ketamine (75 mg/kg bw) and were then sacrificed by euthanasia to allow for tissue dissection.
Sample collection
Male Wistar rats (weight: 160–210 g) were housed under standard conditions (25°C ± 2°C, humidity 60% ± 5%, 12/12-h light/dark cycle) for a period of 2 weeks. Animals received standard extruded chow and water ad libitum until either treatment or sacrifice. All experimental procedures followed the guidelines proposed by the Brazilian Society of Laboratory Animal Science and were approved by the Ethics Committee for Animal Research of the Federal University of Uberlândia, Brazil (CEUA/UFU 051/08).
The hearts were quickly removed, washed with chilled 0.9% NaCl, weighed and either immersed in liquid nitrogen or fixed in 10% formaldehyde solution in 0.1 M phosphate-buffered saline (pH 7.4). The ratio of heart weight (mg) to body weight (g) was calculated. Blood was collected from the portal vein to measure blood glucose and serum biochemical parameters such as total cholesterol (using the enzymatic Trinder method), plasma triglycerides (using the enzymatic Trinder method), total protein (using the Biuret method), creatinine (using a modified Heinegard and Tiderstram’s method), urea [using the urease ultraviolet (UV) kinetic method], aspartate aminotransferase (AST) [using the International Federation of Clinical Chemistry (IFCC) UV kinetic method], alanine aminotransferase (ALT) (using the IFCC UV kinetic method), γ-glutamyltransferase (γ-GT) (using a modified Szasz method) and alkaline phosphatase (using a modified Bowers and McComb method). All parameters were measured in a clinical analysis laboratory at the Faculty of Veterinary Medicine of the Federal University of Uberlândia with a Cobas Mira automatic analyser (Roche Diagnostic Systems, Basel, Switzerland) at 37°C using commercially available kits (Labtest Diagnostica©, Lagoa Santa, MG, Brazil).
Diabetes induction
Homogenate preparation
Rats were starved for 24 h and were then anesthetized by an intraperitoneal injection of xylazine [10 mg/kg body weight (bw)] and ketamine (75 mg/kg bw). Diabetes was induced by injection of streptozotocin (single dose, 40 mg/ kg bw) in 0.01 M citrate buffer (pH 4.5) (Sigma–Aldrich, St. Louis, MO, USA) via the penile vein (2 mL/kg bw); 10 days after diabetes induction, the fasting blood glucose level was monitored using reactive strips (Biocheck Glucose Test Strip; Bioeasy, Belo Horizonte, MG, Brazil). Rats with a blood glucose level above 200 mg/dL were considered to be diabetic.
Each heart was homogenized separately on ice in homogenization buffer containing 40 mM HEPES, 100 mM ethylenediaminetetraacetic (EDTA), 2 mM ethylene glycol tetraacetic acid (EGTA), 2 mM dithiothreitol (DTT), 1 mM benzamidine and 0.5 mM phenylmethanesulphonylfluoride (PMSF). The homogenates were centrifuged at 10,000g for 10 min at 4°C, and the total protein concentration in the supernatants was measured by the Bradford14 assay. Supernatants were assayed for the activity of oxidative stress markers.
Methods Animals
Analysis of oxidative stress markers
Groups and treatments Rats were randomly divided into six groups (n = 8 rats/ group): non-diabetic (ND); non-treated diabetic (NTD); diabetic treated with 25 mg/kg acarbose (DACA) and diabetic treated with 100, 500 or 1500 mg/kg commercial
The levels of lipid peroxidation in the tissue were determined by measuring malondialdehyde (MDA) by the thiobarbituric acid reactive substances (TBARS) assay using a commercial kit (Cayman Chemical Company, Ann Arbor, MI, USA). The total antioxidant status (TAS) and
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ND: non-diabetic; NTD: non-treated diabetic; D100, D500 and D1500: diabetic rats treated with phaseolamin at 100, 500 and 1500 mg/kg, respectively; DACA: diabetic rats treated with acarbose; γ-GT: γ-glutamyltransferase; AST: aspartate aminotransferase; ALT: alanine aminotransferase. ap < 0.05 versus ND. bp < 0.001 versus ND. cp < 0.05 versus NTD.
284.00 ± 17.59 1047.50 ± 103.30 3.66 ± 0.13 397.66 ± 39.33b 82.68 ± 4.91 0.68 ± 0.07 359.08 ± 82.25a 17.21 ± 10.73 92.00 ± 6.76 78.00 ± 10.94a 117.13 ± 55.54a 61.13 ± 6.11a 301.00 ± 12.83 967.50 ± 35.67a 3.22 ± 0.13 333.57 ± 43.88b,c 71.41 ± 3.46 0.57 ± 0.09 261.65 ± 55.52c 6.20 ± 2.85 100.57 ± 8.94 62.28 ± 6.25 99.72 ± 17.66 48.88 ± 6.21c 270.75 ± 16.54 930.00 ± 25.49a 3.47 ± 0.25 415.40 ± 53.48b,c 65.60 ± 5.91 0.68 ± 0.66 319.18 ± 77.68 9.08 ± 3.42 120.80 ± 22.96 64.40 ± 15.66 125.40 ± 13.42 54.54 ± 4.41c Body weight (g) Heart weight (mg) Heart/body weight Glycaemia (mg/dL) Cholesterol (mg/dL) Creatinine (mg/dL) Alkaline phosphatase (U/L) γ-GT (U/L) AST (U/L) ALT (U/L) Triglycerides Urea (mg/dL)
319.75 ± 6.90 1305.00 ± 130.41 4.10 ± 0.46 104.12 ± 5.32 65.33 ± 4.99 0.62 ± 0.03 92.41 ± 7.45 5.38 ± 1.42 74.62 ± 3.52 36.37 ± 2.30 59.96 ± 9.57 37.26 ± 1.68
255.00 ± 16.29a 987.50 ± 20.15 3.97 ± 0.30 568.57 ± 38.04a 70.45 ± 4.72 0.87 ± 0.05 559.32 ± 62.14b 19.80 ± 9.35 116.71 ± 11.38 97.71 ± 6.19b 98.93 ± 40.60 77.68 ± 5.59b
233.00 ± 12.34a 877.50 ± 47.67b 3.79 ± 0.26 330.00 ± 85.68b,c 65.90 ± 2.09 0.66 ± 0.07 318.16 ± 19.77 10.11 ± 5.86 93.33 ± 10.84 76.50 ± 10.59a 114.53 ± 18.45 57.03 ± 5.22
DACA D500
D1500
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D100 NTD ND
Table 1. Body and heart weight, and biochemical parameters.
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superoxide dismutase (SOD) activity were measured using a commercial kit (Randox Laboratories Ltd, Crumlin, UK). Catalase (CAT) activity was assayed spectrophotometrically by monitoring hydrogen peroxide decomposition at 240 nm,15 with a substrate concentration of 20 mM for cardiac tissue measurements. All analyses were performed in duplicate, and samples were normalized to protein concentration.
Histology and measurement of collagen Heart tissue was fixed with 10% formaldehyde in 0.1 M phosphate-buffered saline (pH 7.4) for 24 h, dehydrated in ethanol, cleared in xylene and embedded in paraffin. To determine the percentage of connective tissue, 5-µm sections were stained with picrosirius red.16,17 Picrosirius red technique is used to analyse collagen content because it binds to collagen molecules other than type I, such as type III.18 Morphometric analysis was carried out using a light microscope (Olympus Biotech UK Ltd, Hertfordshire, UK) with a 40× objective, and the microscope was equipped with an Oly-200 charge-coupled device (CCD) camera linked to a personal computer (PC) with a captureand-image-analysis system (HL-Image 97; Western Vision Software, Layton, UT, USA). In total, 30 random fields within areas that had a higher proportion of staining were selected, and the percentage of collagen pixels per field area was measured.
Statistical analysis All the values are expressed as the mean ± standard error of mean (SEM). Data were analysed by one-way analysis of variance (ANOVA) using SigmaStat 3.5 software (Systat Software, Inc., Chicago, IL, USA); p < 0.05 was considered to be statistically significant.
Results Table 1 summarizes the mean changes in body and cardiac weight, as well as biochemical parameters such as glycaemia, cholesterol, creatinine, alkaline phosphatase, γ-GT, AST, ALT, triglycerides and urea. After 20 days, the NTD group presented lower body weight and increased glycaemia, alkaline phosphatase, ALT and urea levels compared to the ND group. Treatment with phaseolamin decreased the blood glucose of diabetic animals by 26% and 42% (D100 and D500 groups, respectively), and administration of acarbose caused a 30% glycaemic reduction. The serum activity of alkaline phosphatase was fivefold higher in the NTD group compared to the ND group. Groups of diabetic animals treated with phaseolamin showed a 44% and 54% reduction in enzyme activity (D100 and D1500 groups, respectively), and the DACA group showed a 36% reduction in the enzyme activity compared to the NTD group.
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Figure 1. The effect of diabetes and phaseolamin treatment on oxidative stress biomarker levels in rat hearts. (a) Total antioxidant activity: ‘a’ represents p < 0.001 versus other groups; ‘b’ represents p < 0.001 versus other groups. (b) Superoxide dismutase activity: ‘a’ represents p < 0.001 versus ND, D100, D500 and DACA. (c) Malondialdehyde concentration: ‘a’ represents p < 0.001 versus NTD, D100, D500 and DACA; ‘b’ represents p < 0.001 versus NTD, D100 and DACA. (d) Catalase activity: ‘a’ represents p < 0.001 versus other groups.
ND: non-diabetic; NTD; non-treated diabetic; D100, D500 and D1500: diabetic animals supplemented with phaseolamin at 100, 500 and 1500 mg/kg, respectively; DACA: diabetic treated with 25 mg/kg acarbose; MDA: malondialdehyde; SEM: standard error of mean. Bars represent the mean ± SEM.
The urea level was twofold higher in the NTD group compared to the ND group. The D500 and D1500 groups that were treated with phaseolamin had a reduced serum concentration of urea compared to the NTD group. No changes were observed in the cholesterol, creatine and AST levels. Enzymes involved in the antioxidant defence system were impaired in the hyperglycaemic state, resulting in a reduction of the total antioxidant activity of the NTD group compared to the ND group. However, treatment with acarbose and phaseolamin caused an increase in the TAS levels in cardiac tissue compared to the NTD group (Figure 1(a)). In contrast to the lack of an effect on the enzymatic defence system, TAS was negatively correlated to MDA concentration (r = −0.7485), CAT activity (r = −0.6545), sulfhydryl group linked to protein (r = −0.6371) and collagen content (r = −0.6322).
Under hyperglycaemic conditions, SOD activity was higher in the NTD group compared to the ND group (Figure 1(b)). Groups treated with either acarbose or phaseolamin (D100, D500 and DACA) had decreased SOD activity compared to the NTD group. We observed an increase in CAT activity in the NTD group compared to the ND group and the other diabetic groups that were treated (Figure 1(d)). There was no difference between diabetic animals that were treated (D100, D500, D1500 and DACA) compared to the ND group. However, correlation analysis showed that hyperglycaemia is positively correlated to CAT activity (r = 0.67). After induction of diabetes, the marker of lipid peroxidation products (MDA) was increased in the NTD, D100, D500 and DACA groups compared to the ND group. However, the MDA concentration of the D1500 group was similar to that of the ND group (Figure 1(c)).
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Figure 2. The deposition of collagen fibres in rat hearts. (a) Total content of collagen: *p < 0.001 versus NTD; **p < 0.05 versus NTD. (b) Histological sections of rat heart stained with picrosirius red.
ND: non-diabetic; NTD; non-treated diabetic; D100, D500 and D1500: diabetic animals supplemented with phaseolamin at 100, 500 and 1500 mg/kg, respectively; DACA: diabetic treated with 25 mg/kg acarbose; SEM: standard error of mean. Bars represent the mean ± SEM.
The total content of collagen was examined using the picrosirius red method (Figure 2). We observed that there was increased collagen deposition in the rat hearts from the NTD group. The D100 and D500 groups did not display any decrease in collagen content and had higher collagen concentrations than the ND group and similar concentrations to the NTD group. The D1500 and DACA groups showed reduced values of collagen compared to the NTD group, and no difference was observed compared to the ND group (Figure 2(a)). Groups treated with phaseolamin (D500, D1500 and DACA) displayed attenuation of the weight loss caused by diabetes, and these groups showed a strong negative correlation between weight and collagen deposition in the cardiac tissue (r = −0.80).
Discussion The increased oxidative stress in diabetes due to the overproduction of ROS may be related to an increase in collagen and the development of cardiovascular diseases such as diastolic stiffness.19,20 Phaseolamin, an inhibitor of the alpha-amylase enzyme extracted from white beans, has become more popular in the weight management industry and has been linked to a reduced risk of diabetes, obesity,21 coronary heart disease,22 colon cancer23 and gastrointestinal disorders.24 Our results revealed a marked weight decrease in diabetic animals, but no changes in the heart/body weight ratio were observed in these animals. The blood glucose of diabetic animals reached high levels; however, phaseolamin treatment was able to reduce glycaemia after 20 days. Phaseolamin has a mechanism similar to acarbose, which is based on delayed and/or prevented digestion of carbohydrates due to competitive inhibition of glucosidases.25 Furthermore, significant weight loss was observed after 20
days of streptozotocin induction with the exception of those treated with phaseolamin. Other studies have verified a reduction of weight at 3,26 2127 and 60 days28 after streptozotocin induction. The liver enzymes ALT and AST reveal possible liver damage. ALT is also a more specific indicator of liver inflammation and contributes to the cleavage of protein cross-linkage with collagen, thus reducing the stiffness of arteries and the heart.29 Low serum ALT levels were observed in rats treated with phaseolamin. It is possible that this treatment attenuates the hyperglycaemic condition and stiffness in the heart caused by cross-linking between collagen. Creatinine and urea were used as markers for renal injuries. Creatinine remained stable in both treated and nontreated rats, indicating preserved renal function. However, elevated urea levels were observed in diabetic rats, which may indicate initial damage caused by the hyperglycaemic state. Relative to oxidative stress, phaseolamin treatment increased total antioxidant activity and promoted a compensatory mechanism that reduced antioxidant enzyme function. Moreover, this treatment was able to attenuate lipid peroxidation in diabetic rats. CAT activity has been previously found to be elevated in several tissues and organs of diabetic rats, including the heart,30–33 aorta,30,34 liver,35 parotid glands,36 kidney and brain.37 This alteration of CAT activity is due to diabetes and may be normalized by treatments such as captopril, aminoguanidine,38 acetylsalicylic acid35 and dehydroepiandrosterone.37 However, 20 days of either phaseolamin or acarbose treatment was sufficient to reduce the cardiac CAT activity. It is possible that this downregulation generates an anti-hyperglycaemic effect that minimizes the ROS levels in the hearts of the rats. However, the effects of CAT are still somewhat
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Oliveira et al. conflicting because a recent study showed a protective role of overexpressed CAT that prevented the development of diseases such as myocyte hypertrophy, apoptosis and interstitial fibrosis.39 Distinct SOD activity had been observed in cardiac tissue: decreased SOD activity at 4 and 8 weeks after induction of diabetes40 and increased SOD activity at 32 weeks.41 Despite the fact that diabetes leads to increased SOD levels due to the increased dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide as an adaptive response to oxidative stress, phaseolamin and acarbose treatments reduce its activity. Streptozotocin-induced diabetes regularly results in an increase in MDA,30,42,43 which indicates indirect damage caused by free radicals and is associated with the onset of myocardial fibrosis.44 Phaseolamin treatment decreases cardiac MDA concentrations to levels similar to those of NDs. This antioxidant activity may be associated with phenolic compounds present in beans, either in isolation or in combination with other components that have antioxidant action.45 Histopathological examination showed a marked and significant increase in collagen deposition in diabetic rats as well as a reduction in this deposition in diabetic rats that received phaseolamin treatment, confirming its cardioprotective action. Recent reports have demonstrated the protective action of acarbose, showing beneficial effects on endothelial function,46 anti-hyperglycaemic action47 and cardioprotective effects.48 The hyperglycaemic state is the primary driver of the formation of AGEs49 and has a key role in the development of diabetes-related complications5 that affect the structure and function of the myocardium. These complications are generally attributed to increased collagen deposition in this tissue.50 Studies have shown that glucose plays an important role in the accumulation of collagen in tissues through the increase in collagen synthesis at the transcriptional level.51,52 The glycation process of collagen inhibits its degradation by interfering with the action of metalloproteinases53 and protein kinases activated by mitogen to further myocardial interstitial fibrosis.49,54 AGE formation modifies proteins by forming links between the amino groups and other proteins, which alters the structure of the extracellular matrix and leads to myocardial stiffness.53 CAT overexpression preserves cardiac morphology, prevents changes in contractile function and reduces the concentrations of MDA and AGEs.55 We showed increased activity of antioxidant enzymes (SOD and CAT) as well as lipid peroxidation in diabetic rats, but the increased activity of these enzymes alone was insufficient to reduce oxidative stress and collagen deposition in the heart. However, phaseolamin treatment attenuated the action of ROS and reduced the collagen content in the heart. This effect may be a result of decrease in AGEs’ concentration because glycation causes increased synthesis of various types of collagens.56
In summary, the short-term anti-hyperglycaemic effect of phaseolamin treatment may prevent initial changes caused by oxidative stress and the deposition of collagen as well as reduce the occurrence of heart complications. However, additional studies should be carried out in detail to evaluate any long-term clinical changes. Acknowledgements The authors thank the School of Veterinary Medicine, Federal University of Uberlândia; Prof. Dr Antonio Vicente Mundim and Felipe Cesar Gonçalves for their help in processing the biochemical analyses. The authors also thank Neire Moura de Gouveia, Fernanda Vieira Alves and Fabiana Barcelos Furtado for the technical support.
Declaration of conflicting interests The authors declare that there are no conflicts of interest.
Funding This study was funded by the Brazilian Governmental Agencies from Ministry of Health (Research Program for SUS, PPSUS) and FAPEMIG (#EDT-3257/06). RJSO, SRD and LKC received fellowship from CAPES, and FSE is a CNPq fellow.
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