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Protective effect of glycine in streptozotocin-induced diabetic cataract through aldose reductase inhibitory activity
Biomedicine & Pharmacotherapy 114 (2019) 108794
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Protective effect of glycine in streptozotocin-induced diabetic cataract through aldose reductase inhibitory activity Wei Lia,1, Yujie Zhangb,1, Na Shaoa, a b
T
⁎
Department of Ophthalmology, Jining First People’s Hospital, Jining, Shandong, China Department of Ophthalmology, Affiliated Hospital of Jining Medical University, Jining, Shandong, 272000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Glycine Diabetes Aldose reductase Glucose Insulin
Glycine is a proteinogenic amino acid that serves as a precursor for several proteins. The anti-cataract effects of lysine and other amino acid mixtures in animal models have been reported. Normal rats were administered saline and formed the normal control group (group I). Diabetic rats were administered streptozotocin and were the diabetic control group (group II). Rats were administered glycine (250 mg and 500 mg/kg of body weight) formed groups III and IV, respectively. Diabetic rats were administered sorbinil and were served as positive control (group V). The body weight changes, serum glucose, plasma insulin, total protein, glutathione (GSH) content, and mRNA and protein levels of aldose reductase were determined. Glycine treatment increased body weight gain, reduced blood glucose, and increased plasma insulin levels compared to diabetic control rats, and also increased GSH content and decreased mRNA and protein levels of aldose reductase compared to their respective controls. In summary, glycine supplementation effectively inhibited aldose reductase enzyme activity in experimental diabetic rats.
1. Introduction Diabetes is a chronic metabolic disorder that affects millions of patients worldwide. Diabetes occurs due to insufficient insulin levels and insulin resistance [1–3]. Diabetic cataract is a frequent cause of blindness [4]. In developing countries, diabetic cataract occurs mainly in younger patients [5]. In the polyol pathway, the enzyme aldose reductase plays a critical role in hyperglycemia [6]. Investigators have reported that glucose is converted into sorbitol by aldose reductase in the ocular lens, and sorbitol accumulation leads to swelling in the eye due to higher osmotic pressure [7]. The success of therapeutic approaches to diabetic cataract, therefore, depends on aldose reductase inhibition in lens epithelial tissues. Zhu [8] reported that epalrestat, fidarestat, ranirestat, zenarestat, sorbinil, ranirestat, and tolrestat are all aldose reductase inhibitors. Schemmel et al. [9] reported adverse pharmacokinetic events and unacceptable side effects in association with the use of these aldose reductase inhibitors. Thus, the development of new aldose reductase inhibitors without, or at least with fewer, adverse effects is required for more efficacious treatment to diabetic cataract. Glycine is a proteinogenic amino acid and serves as a precursor for several proteins [10]. The oral medium lethal dose (LD50) of glycine in
rats is 7930 mg/kg [10]. Investigators have reported that glycine serves as an inhibitory neurotransmitter in the brain stem, spinal cord, central nervous system, and retina [10]. Ahmed and Thornalley [11] reported the development of a diabetic cataract due to glycation of several important proteins, which led to cross-linking and conformational changes of the proteins. Other investigators have reported the reduced glycation in lens proteins in the presence of amino acids [12]. Sulochana et al. [13] observed the anti-cataract effect of lysine and other amino acid mixtures in an animal model. However, there has been no report on the effect of glycine on diabetic cataracts. The present study, therefore, evaluated the therapeutic efficacy of glycine against cataract development in streptozotocin diabetic male albino rats. 2. Materials and methods 2.1. Materials Streptozotocin (CAS number: 18883-66-4; molecular weight: 265.22; catalogue number: S0130), sorbinil (S7701-25MG) and glycine (CAS number: 56-40-6; molecular weight: 75.07; catalogue number: G8898) were purchased from Sigma-Aldrich (Shanghai, China).
⁎
Corresponding author at: Department of Ophthalmology, Jining First People’s Hospital, No. 6 of Health Road, Jining, Shandong, China. E-mail address: [email protected] (N. Shao). 1 These two authors contribute to this work equally. https://doi.org/10.1016/j.biopha.2019.108794 Received 7 January 2019; Received in revised form 14 March 2019; Accepted 14 March 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Biomedicine & Pharmacotherapy 114 (2019) 108794
W. Li, et al.
2.2. Rats
2.8. Determination of glutathione content
Male albino rats (160–180 g) were obtained from the animal house of Jining First People's Hospital, Jining Shi, China. Animals were kept in an animal house at room temperature with a relative humidity of 62 ± 5%, a photoperiod of 12 h light and 12 h dark, and free access to food and water. All the animals were handled according to the internationally accepted ethical committee procedures (Medical Ethics Committee of Jining First People's Hospital, 2019-18).
Glutathione (GSH) content was determined in the rat lens tissue homogenate according to a previously reported method [17] based on Ellman’s reaction. Briefly, the sulfhydryl group of GSH reacted with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and the final absorbance was measured at 412 nm. 2.9. Determination of aldose reductase Aldose reductase was determined in lens tissue homogenates according to a previously reported method. The kinetic properties of aldose reductase, i.e., the maximum velocity (Vmax), Michaelis constant (Km), and inhibition constant (Ki) was also assessed based on the Cheng-Prusoff equation [18].
2.3. Experimental diabetes Streptozotocin was used for the induction of experimental diabetes. Briefly, streptozotocin (60 mg/kg body weight) was dissolved in 0.1 M citrate buffer, pH 4.5. The dose was adjusted to a concentration of 1 ml and administered intraperitoneally. Blood glucose was significantly higher in rats subject to experimental diabetes induction compared to normal rats at the end of 72 h [2].
2.10. Reverse transcription-polymerase chain reaction (RT-PCR) RNA was extracted from the lens tissues of rats. Then, oligo primers were used to convert RNA into cDNA. RT-PCR was carried out on aldose reductase with the following primers: forward, 5′-GGACCTCTACCTTA TTCACTG-3′ and reverse, 5′-TTGGCCCAGGGCCTGTCAG-3′. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a PCR internal control (forward, 5′-GGTCACCAGGGCTGCTTTT-3′ and reverse, 5′-ATCTCGCTCCTGGAAGATGGT-3′). The mRNA expression level of aldose reductase was determined based on the 2−△△CT method [19].
2.4. Treatment Glycine was dissolved in normal saline and administered through oral gavage for 45 consecutive days. The experimental animals were assigned to the following groups. Normal rats administered saline served as the normal controls (group I). Diabetic rats administered normal saline served as the diabetic controls (group II). Rats were administered 250, and 500 mg glycine/kg of body weight formed groups III and IV, respectively. Diabetic rats were administered sorbinil (25 mg/kg) and were the positive control group (group V).
2.11. Immunohistochemistry Lens tissues were dissected and fixed in formalin. The tissues were then embedded in paraffin and sections were deparaffinized. Graded alcohol and xylene were used for rehydration. Hydrogen peroxide (0.3%) was used for the inhibition of endogenous peroxide activity. Nonspecific binding sites were blocked by incubation with 2% bovine serum albumin for 30 min. Primary antibody to aldose reductase was then incubated, first with lens sections for 12 h at a cold temperature and then with fluorescein isothiocyanate-conjugated secondary antibody for 60 min. The sections were then viewed under a fluorescence microscope, and the staining intensity was calculated [20].
2.5. Preparation of lens tissue homogenates At the end of treatment, the rats were sacrificed by cervical dislocation, and the eyeballs were removed immediately for the preparation of a homogenate. The lens tissues were carefully washed, and weights were recorded. The 10% homogenate was prepared in phosphate-buffered saline (PBS) and centrifuged at 10,000×g for 10 min. The supernatant was collected for further biochemical assays [3].
2.12. Statistical analysis
2.6. Determination of body weight, serum glucose, and plasma insulin
Experimental values are presented as means ± standard deviation (SD). Differences between the control and treated groups were analyzed using Student’s t-test and analysis of variance (SPSS 17, IBM SPSS Statistics, Hong Kong). A value of P < 0.05 was considered significant.
Rat body weight changes were recorded in all groups. The glucose oxidase method was used for the determination of blood glucose, which was based on the conversion of glucose into gluconic acid [14]. Briefly, 1.8 ml of sodium sulfate-zinc sulphate and 0.1 ml of 2 N Sodium hydroxide were added to the centrifuge tube. Then, 0.1 ml of blood was added to the centrifuge tube and centrifuged at 2000 rpm for 10 min, and 0.5 ml of supernatant was collected in a fresh tube. Then, 5 ml of glucose oxidase solution was added to the same tube and incubated for 60 min at room temperature. The concentration of insulin in plasma samples was determined using Enzyme-Linked Immunoabsorbent Assay (ELISA) method using insulin kit (ab200011, Abcam). The non-hemolysed plasma was used for insulin determination. After a standard procedure, the standard curve was plotted, and the level of insulin in the blood plasma was estimated through interpolation from the standard curve [15].
3. Results The protective effect of glycine against cataractogenesis via inhibition of aldose reductase activity was determined in streptozotocin-induced diabetic male albino rats. Glycine treatment increased body weight gain compared to diabetic controls. The body weight gain was 52% and 63.6% in 250 mg/kg and 500 mg/kg of glycine treatment, respectively, while body weight gain was 64.8% in the positive control (P < 0.05; Table 1). Glycine treatment increased reduced blood glucose by 31.7% and 89.5% in 250 mg/kg and 500 mg/kg of glycine treatment, respectively, while blood glucose was reduced 63.1% in the positive control (P < 0.05; Table 1). Plasma insulin content was increased by 105.2% and 151.1% in 250 mg/kg and 500 mg/kg of glycine treatment respectively, while plasma insulin was increased 197.8% in the positive control (P < 0.05; Table 1). Glycine treatment significantly increased the GSH content in the lens tissue of diabetic rats compared to their respective controls. The GSH content was significantly increased, by 90.9% and 254.5%, in
2.7. Determination of total protein Total protein in the lens tissues was determined according to a previous method [16]. Briefly, the lens proteins reacted with FolinCiocalteau reagent yield a blue color, which was measured at 680 nm (UV-VIS-NIR Spectrophotometer, Shimadzu, Shangai, China). 2
Biomedicine & Pharmacotherapy 114 (2019) 108794
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Table 1 Effect of glycine on body weight, blood glucose and insulin in normal and diabetic male albino rats. Experimental groups
Body weight (g) Initial
Group Group Group Group Group *
I II III IV V
171 168 175 176 179
± ± ± ± ±
Blood glucose (mmol/l) Final
5.7 5.5 6 5.5 6.3
323 205 266 288 295
± ± ± ± ±
11.7 10* 9.6a 6.5a 8.4a
Blood insulin (microunit/ml)
Initial
Final
Initial
Final
5.1 ± 0.2 21.5 ± 1.2* 24.3 ± 1.0 23.5 ± 1.6 22.2 ± 1.5
5.2 ± 0.3 25.4 ± 1.5* 16.6 ± 1.3a 12.4 ± 1.2a 8.2 ± 0.9a
15.3 ± 0.3 4.7 ± 0.3* 4.2 ± 0.3 4.3 ± 0.2 4.5 ± 0.2
15.6 ± 0.4 4.6 ± 0.3* 8.8 ± 0.4a 10.8 ± 0.4a 13.4 ± 0.7a
P < 0.05 (group II vs group I) and aP < 0.05 (group III, IV and V vs group II). Fig. 1. Determination of effect of glycine on glutathione (GSH) levels in the experimental rats. Values are expressed as nmol/ mg of tissue. Experimental results are presented as the mean with SD (n = 6). Statistical analysis was carried out with analysis of variance (ANOVA). *P < 0.05 (group II vs. group I) and aP < 0.05 (groups III, IV and V vs. group II).
Fig. 2. Determination of effect of glycine on aldose reductase activity in the experimental rats. Values are expressed as a percentage of inhibition. Experimental results are presented as the mean with SD (n = 6). Statistical analysis was carried out with analysis of variance (ANOVA). *P < 0.05 (group II vs. group I) and aP < 0.05 (groups III, IV and V vs. group II).
500 mg/kg of glycine, respectively, while aldose reductase enzyme inhibition was 366.5% in the positive control (P < 0.05, Fig. 2). The kinetic properties of aldose reductase were determined following glycine supplementation. The Vmax, Km, and Ki of aldose reductase were determined in control and glycine-treated diabetic rats and showed significantly lower values in the lens tissue of diabetic rats. Following glycine supplementation, the Vmax of aldose reductase was 0.21833 and
250 mg/kg and 500 mg/kg of glycine treatment, respectively, while GSH content was increased 272.7% in the positive control (P < 0.05; Fig. 1). Aldose reductase activity is expressed as a percentage of inhibition. Aldose reductase enzyme activity was significantly inhibited following glycine supplementation compared to their respective controls. The percentage of aldose reductase enzyme inhibition was 99.7%, 18.5%, 36.2% and 77.5% in control, diabetic control, 250 mg/kg and 3
Biomedicine & Pharmacotherapy 114 (2019) 108794
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Table 2 Effect of glycine on kinetic parameters of aldose reductase in normal and diabetic male albino rats.
*
Experimental groups
Vmax
Km × 10−3 mM
Ki
Group Group Group Group Group
0.16264 ± 0.0003 0.43844 ± 0.0005* 0.21833 ± 0.0011a 0.173734 ± 0.0040a 0.168726 ± 0.0005a
0.61245 3.35355 1.94235 0.84524 0.73426
0.00000 2.34325 1.26247 0.74524 0.43326
I II III IV V
± ± ± ± ±
0.0025 0.0245* 0.0426a 0.0011a 0.0004a
± ± ± ± ±
0.0000 0.0215* 0.0212a 0.0032a 0.0005a
P < 0.05 (group II vs group I) and aP < 0.05 (group III, IV and V vs group II).
sorbitol by aldose reductase in the ocular lens; sorbitol accumulation then leads to swelling in the eye due to higher osmotic pressure [7]. The success of diabetic cataract therapy, therefore, depends on aldose reductase inhibition in the lens epithelial tissues. Epalrestat, fidarestat, ranirestat, zenarestat, sorbinil, ranirestat, and tolrestat are well-known aldose reductase inhibitors [8]. However, aldose reductase inhibitors could cause pharmacokinetic events and unacceptable side effects [9]. Anuradha et al. [25] have reported that the amino acids are effective against diabetes and its complications. Several researchers have reported the antioxidant properties of glycine and its role in the inhibition of glycation [26,27]. Sulochana et al. [13] demonstrated the anti-cataract effect of lysine and other amino acid mixtures in animal models. Thus, we used glycine to prevent cataract in streptozotocin diabetic male albino rats in our study. Our results indicate no major differences between normal rats and normal rats treated with glycine. Therefore, no toxic effect of glycine observed on the normal rats, and even the treatment of glycine in diabetic rats reduced the mortality rate. In addition, several researchers have reported that the safe dose of glycine [26,27]. In addition, our experimental results indicate the reduction of blood glucose in diabetic rats following 45 days of glycine supplementation. This agrees with previous findings of Alvarado-Vásquez et al. [26] demonstrating the reduction of glucose following oral administration of glycine. Researchers have reported that the glycine supplementation enhances the response to insulin, and oral supplementation of glycine increases the gut hormone level, which in-turn potentiates insulin effect on blood glucose level [28,29]. Increased lipid peroxidation and reduced GSH, antioxidant enzymes including SOD, catalase and Gpx are due to increased oxidative stress in diabetic conditions. This agrees with previous findings of Zhang et al. [30] demonstrating the same alteration in diabetic cataract condition. Aldose reductase is associated with several diabetic complications, including retinopathy, cataract,
0.173734 in 250 mg/kg and 500 mg/kg of glycine treatment, respectively, while it was 0.168726 in positive control (P < 0.05, Table 2); the Km of aldose reductase was 1.94235 and 0.84524 × 10−3 mM in 250 mg/kg and 500 mg/kg of glycine treatment, respectively, while it was 0.73426 × 10−3 mM in positive control (P < 0.05, Table 2); and the Ki of aldose reductase was 1.26247 and 0.74524 in 250 mg/kg and 500 mg/kg of glycine treatment, respectively, while it was 0.4326 in positive control (P < 0.05, Table 2). The mRNA expression of aldose reductase was substantially increased in diabetic rats. The mRNA expression of aldose reductase was increased 1.4-fold in diabetic rats compared to normal rats. However, mRNA expression of aldose reductase was significantly reduced following glycine supplementation. The mRNA expression of aldose reductase was reduced by 20.8% and 50% in 250 mg/kg and 500 mg/kg of glycine treatment, respectively, while it was 54.2% in the positive control (P < 0.05, Fig. 3). Immunohistochemical analysis further confirmed the reduction of aldose reductase protein expression, by 36% and 75.6%, respectively, while it was 79.8% in the positive control (P < 0.05; Fig. 4B). 4. Discussion Diabetic cataract is opacity in the lens region which interferes with vision, and it is the most common cause of blindness all over the world [21]. Cataract remained a major health problem and long-term complications of diabetes [22]. Diabetes can be controlled through the use of various hypoglycemic agents, dietary changes, and insulin and islet transplantation [23]. Increased polyol pathway, oxidative stress, and formation of advanced glycation end products (AGEs) are known to play a major role in the pathophysiology of diabetic cataract [24]. In the polyol pathway, aldose reductase plays a critical role in hyperglycemia [24]. Investigators have reported that glucose is converted into
Fig. 3. Determination of effect of glycine on aldose reductase mRNA expression in the experimental rats. Values are expressed as fold changes. Experimental results are presented as the mean with SD (n = 6). Statistical analysis was carried out with analysis of variance (ANOVA). *P < 0.05 (group II vs. group I) and aP < 0.05 (groups III, IV and V vs. group II).
4
Biomedicine & Pharmacotherapy 114 (2019) 108794
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Fig. 4. Determination of effect of glycine on aldose reductase expression in the experimental rats. A. Representative immunofluorescent aldose reductase images. B. Analysis of aldose reductase enzyme expression (%).Experimental results are presented as the mean with SD (n = 6). Statistical analysis was carried out with analysis of variance (ANOVA). *P < 0.05 (group II vs. group I) and aP < 0.05 (groups III, IV and V vs. group II).
neuropathy, microangiopathy, and corneal epitheliopathy. All of these diabetic complications can be prevented by inhibiting aldose reductase [31,32]. Furthermore, hyperglycemia leads to peripheral neuropathy, sorbitol accumulation, and several ocular lesions. Sorbitol-lowering agents reduce the formation of cataracts during diabetic conditions [33]. Moreover, reduction of glucose following glycine supplementation delayed the development of diabetic cataracts. Glycine treatment effectively inhibited aldose reductase activity in experimental diabetic rats.
mellitus, Front. Pharmacol. 3 (2012) 87. [7] A. Del Corso, M. Cappiello, U. Mura, From a dull enzyme to something else: facts and perspectives regarding aldose reductase, Curr. Med. Chem. 15 (2008) 1452–1461. [8] C. Zhu, Aldose reductase inhibitors as potential therapeutic drugs of diabetic complications, in: O.O. Oguntibeju (Ed.), Diabetes Mellitus-Insights and Perspectives, InTech, 2013, pp. 17–46. [9] K. Schemmel, R. Padiyara, J. D’souza, Aldose reductase inhibitors in the treatment of diabetic peripheral neuropathy: a review, J. Diabetes Compl. 24 (2010) 354–360. [10] Y. Shibui, T. Miwa, M. Yamashita, K. Chin, T. Kodama, A 4-week repeated dose toxicity study of Glycine in rats by gavage administration, J. Toxicol. Pathol. 26 (2013) 405–412. [11] N. Ahmed, P.J. Thornalley, Advanced glycation endproducts: what is their relevance to diabetic complications? Diabetes Obes. Metab. 9 (2007) 233–245. [12] N. Pescosolido, A. Barbato, R. Giannotti, C. Komaiha, F. Lenarduzzi, Age-related changes in the kinetics of human lenses: prevention of the cataract, Int. J. Ophthalmol. 9 (2016) 1506–1517. [13] K.N. Sulochana, R. Punitham, S. Ramakrishnan, Beneficial effect of lysine and amino acids on cataractogenesis in experimental diabetes through possible antiglycation of lens proteins, Exp. Eye Res. 67 (1998) 597–601. [14] Z. Wang, Y. Yang, X. Xiang, Y. Zhu, J. Men, M. He, Estimation of the normal range of blood glucose in rats, J. Hygeine Res. 39 (2010) 133–137. [15] G.R. Gandhi, P. Sasikumar, Antidiabetic effect of Merremia emarginata Burm. F. in streptozotocin induced diabetic rats, Asian Pac. J. Trop. Biomed. 2 (2012) 281–286. [16] R.A. Warmack, E. Mansilla, R.G. Goya, S.G. Clarke, Racemized and isomerized proteins in aging rat teeth and eye lens, Rejuvenation Res. 19 (2016) 309–317. [17] T. Holm, M.R. Brøgger-Jensen, L. Johnson, L. Kessel, Glutathione preservation during storage of rat lenses in optical-GS and castor oil, PLoS One 8 (2013) e79620. [18] R. Daniellou, H. Zheng, D.R.J. Palmer, Kinetics of the reaction catalyzed by inositol dehydrogenase from Bacillus subtilis and inhibition by fluorinated substrate analogs, Can. J. Chem. 84 (2006) 522–527. [19] M.M. Soliman, M. Abdo Nassan, T.A. Ismail, Origanum Majoranum extract modulates gene expression, hepatic and renal changes in a rat model of type 2 diabetes, Iran. J. Pharm. Res. 15 (2016) 45–54. [20] M.N. Evilsizor, H.F. Ray-Jones, J. Lifshitz, J. Ziebell, Primer for immunohistochemistry on cryosectioned rat brain tissue: example staining for microglia and neurons, J. Vis. Exp. 12 (20159) (2019) e52293. [21] F. Bahmani, S.Z. Bathaie, S.J. Aldavood, A. Ghahghaei, Glycine therapy inhibits the progression of cataract in streptozotocin-induced diabetic rats, Mol. Vis. 18 (2012) 439–448. [22] V.J. Vieira-Potter, D. Karamichos, D.J. Lee, Ocular complications of diabetes and therapeutic approaches, Biomed Res. Int. (2016) (2016) 3801570.
Funding The project was funded by a grant for scientific research projects conducted in Jining (No. 2016-56-31). Conflicts of interest The authors declare that they have no conflict of interest. References [1] P. Muthuraman, D.H. Kim, Therapeutic potential of cyanobacteria against streptozotocin-induced diabetic rats, 3 Biotech 6 (2016) 94. [2] P. Muthuraman, K. Srikumar, A comparative study on the effect of homobrassinolide and gibberellic acid on lipid peroxidation and antioxidant status in normal and diabetic rats, J. Enzyme Inhib. Med. Chem. 24 (2009) 1122–1127. [3] P. Muthuraman, R. Senthilkumar, K. Srikumar, Alterations in beta-islets of Langerhans in alloxan-induced diabetic rats by marine Spirulina platensis, J. Enzyme Inhib. Med. Chem. 24 (2009) 1253–1256. [4] A. Pollreisz, U. Schmidt-Erfurth, Diabetic cataract-pathogenesis, epidemiology and treatment, J. Ophthalmol. (2010) (2010) 608751. [5] V.K. Lathika, T.A. Ajith, Association of grade of cataract with duration of diabetes, age and gender in patients with type II diabetes mellitus, Int. J. Adv. Med. 3 (2016) 304–308. [6] W.H. Tang, K.A. Martin, J. Hwa, Aldose reductase, oxidative stress, and diabetic
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type 2 diabetes mellitus patients, Horm. Metab. Res. 33 (2001) 358–360. [29] M.C. Gannon, J.A. Nuttall, F.Q. Nuttall, The metabolic response to ingested glycine, Am. J. Clin. Nutr. 76 (2002) 1302–1307. [30] S. Zhang, F.Y. Chai, H. Yan, Y. Guo, J.J. Harding, Effects of Nacetylcysteine and glutathione ethyl ester drops on streptozotocin-induced diabetic cataract in rats, Mol. Vis. 14 (2008) 862–870. [31] L. Scotti, M.B. Fernandes, E. Muramatsu, K.F.M. Pasqualoto, V.P. Emereciano, L.C. Tavares, Self-organizing maps and volsurf approach to predict aldose reductase inhibition by flavonoid compounds, Rev. Bras. Phcog. 21 (2011) 170–180. [32] G.B. Reddy, A. Satyanarayana, N. Balakrishna, R. Ayyagari, M. Padma, K. Viswanath, J.M. Petrash, Erythrocyte aldose reductase activity and sorbitol levels in diabetic retinopathy, Mol. Vis. 14 (2008) 593–601. [33] N. Mirsky, R. Cohen, A. Eliaz, A. Dovrat, Featured Article: inhibition of diabetic cataract by glucose tolerance factor extracted from yeast, Exp. Biol. Med. (Maywood) 241 (2016) 817–829.
[23] J.J. Marín-Peñalver, I. Martín-Timón, C. Sevillano-Collantes, F.J. Del CañizoGómez, Update on the treatment of type 2 diabetes mellitus, World J. Diabetes 7 (2016) 354–395. [24] V.P. Singh, A. Bali, N. Singh, A.S. Jaggi, Advanced glycation end products and diabetic complications, Korean J. Physiol. Pharmacol. 18 (2014) 1–14. [25] C.V. Anuradha, Amino acid support in the prevention of diabetes and diabetic complications, Curr. Protein Pept. Sci. 10 (2009) 8–17. [26] N. Alvarado-Vásquez, P. Zamudio, E. Ceron, B. Vanda, E. Zenteno, G. CarvajalSandovala, Effect of glycine in streptozotocin-induced diabetic rats, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 134 (92003) (2019) 521–527. [27] N. Alvarado-Vásquez, R. Lascurain, E. Cerón, B. Vanda, G. Carvajal-Sandovala, A. Tapia, J. Guevara, L.F. Montano, E. Zenteno, Oral glycine administration attenuates diabetic complications in streptozotocin-induced diabetic rats, Life Sci. 79 (2006) 225–232. [28] M. González-Ortiz, R. Medina-Santillan, E. Martinez-Abundis, C.R. von Drateln, Effect of glycine on insulin secretion and action in healthy first-degree relatives of
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Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Protective effect of glycine in streptozotocin-induced diabetic cataract through aldose reductase inhibitory activity Wei Lia,1, Yujie Zhangb,1, Na Shaoa, a b
T
⁎
Department of Ophthalmology, Jining First People’s Hospital, Jining, Shandong, China Department of Ophthalmology, Affiliated Hospital of Jining Medical University, Jining, Shandong, 272000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Glycine Diabetes Aldose reductase Glucose Insulin
Glycine is a proteinogenic amino acid that serves as a precursor for several proteins. The anti-cataract effects of lysine and other amino acid mixtures in animal models have been reported. Normal rats were administered saline and formed the normal control group (group I). Diabetic rats were administered streptozotocin and were the diabetic control group (group II). Rats were administered glycine (250 mg and 500 mg/kg of body weight) formed groups III and IV, respectively. Diabetic rats were administered sorbinil and were served as positive control (group V). The body weight changes, serum glucose, plasma insulin, total protein, glutathione (GSH) content, and mRNA and protein levels of aldose reductase were determined. Glycine treatment increased body weight gain, reduced blood glucose, and increased plasma insulin levels compared to diabetic control rats, and also increased GSH content and decreased mRNA and protein levels of aldose reductase compared to their respective controls. In summary, glycine supplementation effectively inhibited aldose reductase enzyme activity in experimental diabetic rats.
1. Introduction Diabetes is a chronic metabolic disorder that affects millions of patients worldwide. Diabetes occurs due to insufficient insulin levels and insulin resistance [1–3]. Diabetic cataract is a frequent cause of blindness [4]. In developing countries, diabetic cataract occurs mainly in younger patients [5]. In the polyol pathway, the enzyme aldose reductase plays a critical role in hyperglycemia [6]. Investigators have reported that glucose is converted into sorbitol by aldose reductase in the ocular lens, and sorbitol accumulation leads to swelling in the eye due to higher osmotic pressure [7]. The success of therapeutic approaches to diabetic cataract, therefore, depends on aldose reductase inhibition in lens epithelial tissues. Zhu [8] reported that epalrestat, fidarestat, ranirestat, zenarestat, sorbinil, ranirestat, and tolrestat are all aldose reductase inhibitors. Schemmel et al. [9] reported adverse pharmacokinetic events and unacceptable side effects in association with the use of these aldose reductase inhibitors. Thus, the development of new aldose reductase inhibitors without, or at least with fewer, adverse effects is required for more efficacious treatment to diabetic cataract. Glycine is a proteinogenic amino acid and serves as a precursor for several proteins [10]. The oral medium lethal dose (LD50) of glycine in
rats is 7930 mg/kg [10]. Investigators have reported that glycine serves as an inhibitory neurotransmitter in the brain stem, spinal cord, central nervous system, and retina [10]. Ahmed and Thornalley [11] reported the development of a diabetic cataract due to glycation of several important proteins, which led to cross-linking and conformational changes of the proteins. Other investigators have reported the reduced glycation in lens proteins in the presence of amino acids [12]. Sulochana et al. [13] observed the anti-cataract effect of lysine and other amino acid mixtures in an animal model. However, there has been no report on the effect of glycine on diabetic cataracts. The present study, therefore, evaluated the therapeutic efficacy of glycine against cataract development in streptozotocin diabetic male albino rats. 2. Materials and methods 2.1. Materials Streptozotocin (CAS number: 18883-66-4; molecular weight: 265.22; catalogue number: S0130), sorbinil (S7701-25MG) and glycine (CAS number: 56-40-6; molecular weight: 75.07; catalogue number: G8898) were purchased from Sigma-Aldrich (Shanghai, China).
⁎
Corresponding author at: Department of Ophthalmology, Jining First People’s Hospital, No. 6 of Health Road, Jining, Shandong, China. E-mail address: [email protected] (N. Shao). 1 These two authors contribute to this work equally. https://doi.org/10.1016/j.biopha.2019.108794 Received 7 January 2019; Received in revised form 14 March 2019; Accepted 14 March 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Biomedicine & Pharmacotherapy 114 (2019) 108794
W. Li, et al.
2.2. Rats
2.8. Determination of glutathione content
Male albino rats (160–180 g) were obtained from the animal house of Jining First People's Hospital, Jining Shi, China. Animals were kept in an animal house at room temperature with a relative humidity of 62 ± 5%, a photoperiod of 12 h light and 12 h dark, and free access to food and water. All the animals were handled according to the internationally accepted ethical committee procedures (Medical Ethics Committee of Jining First People's Hospital, 2019-18).
Glutathione (GSH) content was determined in the rat lens tissue homogenate according to a previously reported method [17] based on Ellman’s reaction. Briefly, the sulfhydryl group of GSH reacted with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and the final absorbance was measured at 412 nm. 2.9. Determination of aldose reductase Aldose reductase was determined in lens tissue homogenates according to a previously reported method. The kinetic properties of aldose reductase, i.e., the maximum velocity (Vmax), Michaelis constant (Km), and inhibition constant (Ki) was also assessed based on the Cheng-Prusoff equation [18].
2.3. Experimental diabetes Streptozotocin was used for the induction of experimental diabetes. Briefly, streptozotocin (60 mg/kg body weight) was dissolved in 0.1 M citrate buffer, pH 4.5. The dose was adjusted to a concentration of 1 ml and administered intraperitoneally. Blood glucose was significantly higher in rats subject to experimental diabetes induction compared to normal rats at the end of 72 h [2].
2.10. Reverse transcription-polymerase chain reaction (RT-PCR) RNA was extracted from the lens tissues of rats. Then, oligo primers were used to convert RNA into cDNA. RT-PCR was carried out on aldose reductase with the following primers: forward, 5′-GGACCTCTACCTTA TTCACTG-3′ and reverse, 5′-TTGGCCCAGGGCCTGTCAG-3′. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a PCR internal control (forward, 5′-GGTCACCAGGGCTGCTTTT-3′ and reverse, 5′-ATCTCGCTCCTGGAAGATGGT-3′). The mRNA expression level of aldose reductase was determined based on the 2−△△CT method [19].
2.4. Treatment Glycine was dissolved in normal saline and administered through oral gavage for 45 consecutive days. The experimental animals were assigned to the following groups. Normal rats administered saline served as the normal controls (group I). Diabetic rats administered normal saline served as the diabetic controls (group II). Rats were administered 250, and 500 mg glycine/kg of body weight formed groups III and IV, respectively. Diabetic rats were administered sorbinil (25 mg/kg) and were the positive control group (group V).
2.11. Immunohistochemistry Lens tissues were dissected and fixed in formalin. The tissues were then embedded in paraffin and sections were deparaffinized. Graded alcohol and xylene were used for rehydration. Hydrogen peroxide (0.3%) was used for the inhibition of endogenous peroxide activity. Nonspecific binding sites were blocked by incubation with 2% bovine serum albumin for 30 min. Primary antibody to aldose reductase was then incubated, first with lens sections for 12 h at a cold temperature and then with fluorescein isothiocyanate-conjugated secondary antibody for 60 min. The sections were then viewed under a fluorescence microscope, and the staining intensity was calculated [20].
2.5. Preparation of lens tissue homogenates At the end of treatment, the rats were sacrificed by cervical dislocation, and the eyeballs were removed immediately for the preparation of a homogenate. The lens tissues were carefully washed, and weights were recorded. The 10% homogenate was prepared in phosphate-buffered saline (PBS) and centrifuged at 10,000×g for 10 min. The supernatant was collected for further biochemical assays [3].
2.12. Statistical analysis
2.6. Determination of body weight, serum glucose, and plasma insulin
Experimental values are presented as means ± standard deviation (SD). Differences between the control and treated groups were analyzed using Student’s t-test and analysis of variance (SPSS 17, IBM SPSS Statistics, Hong Kong). A value of P < 0.05 was considered significant.
Rat body weight changes were recorded in all groups. The glucose oxidase method was used for the determination of blood glucose, which was based on the conversion of glucose into gluconic acid [14]. Briefly, 1.8 ml of sodium sulfate-zinc sulphate and 0.1 ml of 2 N Sodium hydroxide were added to the centrifuge tube. Then, 0.1 ml of blood was added to the centrifuge tube and centrifuged at 2000 rpm for 10 min, and 0.5 ml of supernatant was collected in a fresh tube. Then, 5 ml of glucose oxidase solution was added to the same tube and incubated for 60 min at room temperature. The concentration of insulin in plasma samples was determined using Enzyme-Linked Immunoabsorbent Assay (ELISA) method using insulin kit (ab200011, Abcam). The non-hemolysed plasma was used for insulin determination. After a standard procedure, the standard curve was plotted, and the level of insulin in the blood plasma was estimated through interpolation from the standard curve [15].
3. Results The protective effect of glycine against cataractogenesis via inhibition of aldose reductase activity was determined in streptozotocin-induced diabetic male albino rats. Glycine treatment increased body weight gain compared to diabetic controls. The body weight gain was 52% and 63.6% in 250 mg/kg and 500 mg/kg of glycine treatment, respectively, while body weight gain was 64.8% in the positive control (P < 0.05; Table 1). Glycine treatment increased reduced blood glucose by 31.7% and 89.5% in 250 mg/kg and 500 mg/kg of glycine treatment, respectively, while blood glucose was reduced 63.1% in the positive control (P < 0.05; Table 1). Plasma insulin content was increased by 105.2% and 151.1% in 250 mg/kg and 500 mg/kg of glycine treatment respectively, while plasma insulin was increased 197.8% in the positive control (P < 0.05; Table 1). Glycine treatment significantly increased the GSH content in the lens tissue of diabetic rats compared to their respective controls. The GSH content was significantly increased, by 90.9% and 254.5%, in
2.7. Determination of total protein Total protein in the lens tissues was determined according to a previous method [16]. Briefly, the lens proteins reacted with FolinCiocalteau reagent yield a blue color, which was measured at 680 nm (UV-VIS-NIR Spectrophotometer, Shimadzu, Shangai, China). 2
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Table 1 Effect of glycine on body weight, blood glucose and insulin in normal and diabetic male albino rats. Experimental groups
Body weight (g) Initial
Group Group Group Group Group *
I II III IV V
171 168 175 176 179
± ± ± ± ±
Blood glucose (mmol/l) Final
5.7 5.5 6 5.5 6.3
323 205 266 288 295
± ± ± ± ±
11.7 10* 9.6a 6.5a 8.4a
Blood insulin (microunit/ml)
Initial
Final
Initial
Final
5.1 ± 0.2 21.5 ± 1.2* 24.3 ± 1.0 23.5 ± 1.6 22.2 ± 1.5
5.2 ± 0.3 25.4 ± 1.5* 16.6 ± 1.3a 12.4 ± 1.2a 8.2 ± 0.9a
15.3 ± 0.3 4.7 ± 0.3* 4.2 ± 0.3 4.3 ± 0.2 4.5 ± 0.2
15.6 ± 0.4 4.6 ± 0.3* 8.8 ± 0.4a 10.8 ± 0.4a 13.4 ± 0.7a
P < 0.05 (group II vs group I) and aP < 0.05 (group III, IV and V vs group II). Fig. 1. Determination of effect of glycine on glutathione (GSH) levels in the experimental rats. Values are expressed as nmol/ mg of tissue. Experimental results are presented as the mean with SD (n = 6). Statistical analysis was carried out with analysis of variance (ANOVA). *P < 0.05 (group II vs. group I) and aP < 0.05 (groups III, IV and V vs. group II).
Fig. 2. Determination of effect of glycine on aldose reductase activity in the experimental rats. Values are expressed as a percentage of inhibition. Experimental results are presented as the mean with SD (n = 6). Statistical analysis was carried out with analysis of variance (ANOVA). *P < 0.05 (group II vs. group I) and aP < 0.05 (groups III, IV and V vs. group II).
500 mg/kg of glycine, respectively, while aldose reductase enzyme inhibition was 366.5% in the positive control (P < 0.05, Fig. 2). The kinetic properties of aldose reductase were determined following glycine supplementation. The Vmax, Km, and Ki of aldose reductase were determined in control and glycine-treated diabetic rats and showed significantly lower values in the lens tissue of diabetic rats. Following glycine supplementation, the Vmax of aldose reductase was 0.21833 and
250 mg/kg and 500 mg/kg of glycine treatment, respectively, while GSH content was increased 272.7% in the positive control (P < 0.05; Fig. 1). Aldose reductase activity is expressed as a percentage of inhibition. Aldose reductase enzyme activity was significantly inhibited following glycine supplementation compared to their respective controls. The percentage of aldose reductase enzyme inhibition was 99.7%, 18.5%, 36.2% and 77.5% in control, diabetic control, 250 mg/kg and 3
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Table 2 Effect of glycine on kinetic parameters of aldose reductase in normal and diabetic male albino rats.
*
Experimental groups
Vmax
Km × 10−3 mM
Ki
Group Group Group Group Group
0.16264 ± 0.0003 0.43844 ± 0.0005* 0.21833 ± 0.0011a 0.173734 ± 0.0040a 0.168726 ± 0.0005a
0.61245 3.35355 1.94235 0.84524 0.73426
0.00000 2.34325 1.26247 0.74524 0.43326
I II III IV V
± ± ± ± ±
0.0025 0.0245* 0.0426a 0.0011a 0.0004a
± ± ± ± ±
0.0000 0.0215* 0.0212a 0.0032a 0.0005a
P < 0.05 (group II vs group I) and aP < 0.05 (group III, IV and V vs group II).
sorbitol by aldose reductase in the ocular lens; sorbitol accumulation then leads to swelling in the eye due to higher osmotic pressure [7]. The success of diabetic cataract therapy, therefore, depends on aldose reductase inhibition in the lens epithelial tissues. Epalrestat, fidarestat, ranirestat, zenarestat, sorbinil, ranirestat, and tolrestat are well-known aldose reductase inhibitors [8]. However, aldose reductase inhibitors could cause pharmacokinetic events and unacceptable side effects [9]. Anuradha et al. [25] have reported that the amino acids are effective against diabetes and its complications. Several researchers have reported the antioxidant properties of glycine and its role in the inhibition of glycation [26,27]. Sulochana et al. [13] demonstrated the anti-cataract effect of lysine and other amino acid mixtures in animal models. Thus, we used glycine to prevent cataract in streptozotocin diabetic male albino rats in our study. Our results indicate no major differences between normal rats and normal rats treated with glycine. Therefore, no toxic effect of glycine observed on the normal rats, and even the treatment of glycine in diabetic rats reduced the mortality rate. In addition, several researchers have reported that the safe dose of glycine [26,27]. In addition, our experimental results indicate the reduction of blood glucose in diabetic rats following 45 days of glycine supplementation. This agrees with previous findings of Alvarado-Vásquez et al. [26] demonstrating the reduction of glucose following oral administration of glycine. Researchers have reported that the glycine supplementation enhances the response to insulin, and oral supplementation of glycine increases the gut hormone level, which in-turn potentiates insulin effect on blood glucose level [28,29]. Increased lipid peroxidation and reduced GSH, antioxidant enzymes including SOD, catalase and Gpx are due to increased oxidative stress in diabetic conditions. This agrees with previous findings of Zhang et al. [30] demonstrating the same alteration in diabetic cataract condition. Aldose reductase is associated with several diabetic complications, including retinopathy, cataract,
0.173734 in 250 mg/kg and 500 mg/kg of glycine treatment, respectively, while it was 0.168726 in positive control (P < 0.05, Table 2); the Km of aldose reductase was 1.94235 and 0.84524 × 10−3 mM in 250 mg/kg and 500 mg/kg of glycine treatment, respectively, while it was 0.73426 × 10−3 mM in positive control (P < 0.05, Table 2); and the Ki of aldose reductase was 1.26247 and 0.74524 in 250 mg/kg and 500 mg/kg of glycine treatment, respectively, while it was 0.4326 in positive control (P < 0.05, Table 2). The mRNA expression of aldose reductase was substantially increased in diabetic rats. The mRNA expression of aldose reductase was increased 1.4-fold in diabetic rats compared to normal rats. However, mRNA expression of aldose reductase was significantly reduced following glycine supplementation. The mRNA expression of aldose reductase was reduced by 20.8% and 50% in 250 mg/kg and 500 mg/kg of glycine treatment, respectively, while it was 54.2% in the positive control (P < 0.05, Fig. 3). Immunohistochemical analysis further confirmed the reduction of aldose reductase protein expression, by 36% and 75.6%, respectively, while it was 79.8% in the positive control (P < 0.05; Fig. 4B). 4. Discussion Diabetic cataract is opacity in the lens region which interferes with vision, and it is the most common cause of blindness all over the world [21]. Cataract remained a major health problem and long-term complications of diabetes [22]. Diabetes can be controlled through the use of various hypoglycemic agents, dietary changes, and insulin and islet transplantation [23]. Increased polyol pathway, oxidative stress, and formation of advanced glycation end products (AGEs) are known to play a major role in the pathophysiology of diabetic cataract [24]. In the polyol pathway, aldose reductase plays a critical role in hyperglycemia [24]. Investigators have reported that glucose is converted into
Fig. 3. Determination of effect of glycine on aldose reductase mRNA expression in the experimental rats. Values are expressed as fold changes. Experimental results are presented as the mean with SD (n = 6). Statistical analysis was carried out with analysis of variance (ANOVA). *P < 0.05 (group II vs. group I) and aP < 0.05 (groups III, IV and V vs. group II).
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Fig. 4. Determination of effect of glycine on aldose reductase expression in the experimental rats. A. Representative immunofluorescent aldose reductase images. B. Analysis of aldose reductase enzyme expression (%).Experimental results are presented as the mean with SD (n = 6). Statistical analysis was carried out with analysis of variance (ANOVA). *P < 0.05 (group II vs. group I) and aP < 0.05 (groups III, IV and V vs. group II).
neuropathy, microangiopathy, and corneal epitheliopathy. All of these diabetic complications can be prevented by inhibiting aldose reductase [31,32]. Furthermore, hyperglycemia leads to peripheral neuropathy, sorbitol accumulation, and several ocular lesions. Sorbitol-lowering agents reduce the formation of cataracts during diabetic conditions [33]. Moreover, reduction of glucose following glycine supplementation delayed the development of diabetic cataracts. Glycine treatment effectively inhibited aldose reductase activity in experimental diabetic rats.
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