Research Article Received: 21 September 2014
Revised: 12 December 2014
Accepted article published: 31 December 2014
Published online in Wiley Online Library: 2 February 2015
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7067
Hypoglycaemic effects of functional tri-peptides from silk in differentiated adipocytes and streptozotocin-induced diabetic mice Bok Kyung Han,a† Hyun Jung Lee,b† Hyun-Sun Lee,c Hyung Joo Suhb and Yooheon Parkb* Abstract BACKGROUND: In this study, the tri-peptides Gly-Glu-Tyr (GEY) and Gly-Tyr-Gly (GYG), identified previously as active compounds from the silk peptide E5K6, significantly stimulated basal and insulin-mediated glucose uptake by 3T3-L1 fibroblasts in a dose-dependent manner. RESULTS: Synthetic GEY and GYG peptides at a concentration of 500 𝛍mol L−1 significantly increased glucose transporter type 4 expression by 157% and 239%, respectively. Differentiation of 3T3-L1 cells into adipocytes leads to accumulation of intracellular fat droplets, and GEY and GYG at a concentration of 250 𝛍mol L−1 suppressed this effect by 72% and 75%, respectively. GYG improved glucose tolerance in steptozotocin (STZ)-induced diabetic mice in a dose-dependent manner. CONCLUSION: These results suggest that GYG isolated from E5K6 has anti-diabetic potential and silk waste products containing bioactive peptides could be used to the developments of treatments to lower blood glucose. © 2014 Society of Chemical Industry Keywords: silk peptide E5K6; 3T3-L1 adipocyte; glucose uptake; STZ-induced diabetic mice; alcalase
INTRODUCTION
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Diabetes mellitus (DM) is a major endocrine disorder and a global public health concern, affecting nearly 10% of the world’s population. DM comprises a group of syndromes characterised by hyperglycaemia, altered metabolism of lipids, carbohydrates and proteins, and an increased risk of complications from vascular disease.1 Conventional drugs used for DM treatment present significant limitations such as strict and multiple dosing regimens, high cost, inaccessibility, and adverse effects. Studies of alternative treatments for DM have provided valuable leads for the development of therapeutic strategies. In the last few decades, there has been exponential growth in the field of alternative treatments by natural supplements, owing to their natural origin and diminished side effects.2 Because natural products might be less toxic and present fewer side effects than synthetic compounds,3 they might have therapeutic potential for complex disorders, such as diabetes and associated complications.4 Silk is a type of natural fibre produced by silkworms (Bombyx mori). Since the 1970s, many bioactive peptides derived from food protein hydrolysates have been studied as potential nutraceuticals. Based on this body of work, application of silk peptides in the biotechnological and biomedical fields has received increasing attention.5,6 Silk peptides have been employed as a biomedical suture material and are believed to be safe for humans. Silk J Sci Food Agric 2016; 96: 116–121
peptides consist of peptides of different sizes, which create peptides that are 18–20 amino acids in length and enzymatic degradation results in peptides of specific sizes or compositions presenting diverse bioactivities, such as hypocholesterolaemic, antioxidant, immunoregulatory, anti-tumour, antiviral, and antibacterial effects.7 – 9 Previous studies have reported that silk peptides influence blood glucose, insulin and serum lipids levels in vivo,10 – 12 but the underlying mechanisms mediating these effects were not clear because the active compound was not identified. Previous reports from our laboratory demonstrated that the silk peptide E5K6 reduces plasma glucose levels and improves the lipid profile in C57BL/KsJ-db/db mice, increases glucose uptake via up-regulation
∗
Correspondence to: Yooheon Park, Department of Food and Nutrition, Korea University, Seoul 136–703, Republic of Korea. E-mail: [email protected]
† Bok Kyung Han and Hyun Jung Lee contributed equally to this study. a BK Bio Co. Ltd, Sungnam, 462-819, Republic of Korea b Department of Food and Nutrition, Korea University, Seoul 136-703, Republic of Korea c Food Quality & Safety Department, Agency for Korea National Food Cluster, Gwacheon, 427-806, Republic of Korea
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Figure 1. Chemical structure of the tri-peptides GEY and GYG (After a previously published figure15 ).
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MATERIALS AND METHODS Materials The synthetic peptides GEY and GYG were obtained from Peptron (Daejeon, Korea). 3 T3-L1 fibroblast cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cell culture reagents such as low-glucose Dulbecco’s modified Eagle’s medium (DMEM), high-glucose DMEM, fetal bovine serum (FBS), and antibiotics (100 U mL−1 penicillin and 100 μg mL−1 streptomycin) were purchased from Gibco BRL (Grand Island, NY, USA). 2-Deoxy-D-[1,2-3 H] glucose (2-DG) was purchased from PerkinElmer Life (Boston, MA, USA). 3-Isobutylmethyl xanthine (IBMX), dexamethasone, insulin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St Louis, MO, USA). General chemicals and laboratory equipment were purchased from standard sources.
Conditions of cell culture 3 T3-L1 cells were cultured and induced to differentiate into adipocytes as described previously.22 Pre-adipocytes were cultured in high-glucose DMEM with 10% FBS and 1% penicillin–streptomycin at 37 ∘ C in a humidified atmosphere of 5% CO2 , and cells from passages 3 to 20 were used. Cells plated onto six- or 24-well plates were grown to confluence. Two days after reaching confluence (day 0), cells were induced to differentiate in high-glucose DMEM supplemented with 10% FBS, 1 μg mL−1 insulin, 50 mmol L−1 dexamethasone, and 0.8 mmol L−1 IBMX. Two days following the initiation of cellular differentiation (day 2), dexamethasone and IBMX were withdrawn from the media, whereas insulin was withdrawn from the media 4 days after initiation of differentiation (day 4). The differentiated adipocytes were maintained in high-glucose DMEM and 10% FBS until maturity (days 9–12). On the day of the experiment, each well was examined under a microscope to identify the percentage of undifferentiated cells not resembling adipocytes, which are round cells rich in easily distinguishable fat globules. The wells that were used for experimentation contained little to no pre-adipocyte cells and were easily distinguishable as >90% differentiated overall.
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of glucose transporter type (GLUT) 4 and decreases fat accumulation by up-regulating leptin, thereby exhibiting anti-diabetic effects.13,14 Subsequently, by means of 𝛼-glucosidase inhibition assays and amino acid sequencing, we identified and purified two tri-peptides, Gly-Glu-Tyr (GEY) and Gly-Tyr-Gly (GYG), from E5K6. The structure was elucidated, and we reported that these compounds have an anti-diabetic effect (Fig. 1).15 The composition of fibroin protein is 45.9% Gly, 30.3% Ala, 12.1% Ser, 5.3% Tyr, 1.8% Val and 4.7% of the other 15 amino acids. Most of the sequence exhibits low-complexity and 94% of the sequence is occupied by Gly-X dipeptide motifs, where the probability of X being Ala, Ser and Tyr is 64%, 22% and 10%, respectively.16,17 Insulin-mediated glucose transport to appropriate tissues is essential for maintenance of glucose homeostasis.18 Adipocytes and muscle cells are cells highly sensitive to insulin. In adipocytes, insulin stimulates glucose transport by enhancing the translocation of GLUT4 and, to a lesser extent, of GLUT1 from the cytosol to the plasma membrane.19,20 The classical pathway of insulin signalling, involving glucose transporter translocation to the cell surface, is induced by autophosphorylation of the insulin receptor on multiple tyrosine residues following insulin binding. These events result in the tyrosine phosphorylation of a family of insulin receptor substrate (IRS) proteins and activation of a complex network of downstream signal kinases, such as phosphatidylinositol 3-kinase and the serine/threonine kinase protein kinase B.19,21 In previous work, we reported that E5K6 simultaneously stimulates glucose uptake and suppresses fat accumulation; however, a crude preparation was used in this studies, and the active components remain undetermined. We identified two tri-peptides, GEY and GYG, that inhibit 𝛼-glucosidase activity. In the present study, we evaluated the stimulating effect of the GEY and GYG tri-peptides on glucose uptake and their inhibitory effects on fat accumulation in 3 T3-L1 fibroblasts. Our results demonstrate that the GEY and GYG tri-peptides stimulate glucose transport in adipocytes by inducing the translocation of GLUT1 and GLUT4, mediated by activation of insulin-like signalling pathways. This research clarifies the effects of the anti-hyperglycaemic and anti-hyperlipidaemic activities of silk protein hydrolysates.
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Insulin-stimulated glucose uptake assay in 3T3-L1 adipocyte cells Glucose uptake activity was determined in six-well plates. The effects of silk peptide on insulin-stimulated glucose uptake were determined in 3 T3-L1 fibroblasts induced to differentiate into adipocytes for 9–12 days in high glucose DMEM. Glucose uptake activity was analysed by measuring the uptake of 2-DG.23 Differentiated 3 T3-L1 cells were glucose starved following two washes with low glucose DMEM and incubation with 2 mL of low glucose DMEM containing 10% FBS, at 37 ∘ C for 2 h. After washing with Krebs–Ringer–Hepes (KRH) buffer (128 mmol L−1 NaCl, 4.7 mmol L−1 KCl, 1.25 mmol L−1 CaCl2 , 1.25 mmol L−1 MgSO4 , 10.0 mmol L−1 Na2 HPO4 , 20 mmol L−1 Hepes), the cells were incubated with KRH buffer in the presence of sample, with or without 0.2 nmol L−1 insulin at 37 ∘ C for 30 min. Subsequently, 2-DG and glucose, at a final concentration of 3.7 kBq mL−1 (0.1 μCi mL−1 ) and 1 mmol L−1 , respectively, were added for 15 min. Following addition of cold phosphate buffered saline, the cells were digested with 1% Triton X-100 for 40 min at 37 ∘ C and counted by a scintillation counter (Beckman LS 6000; Beckman, Pasadena, CA, USA).
commercial diet (Samyang, Gumi, Korea) containing the following (g 100 kg−1 of diet): moisture, 80; protein, 230; fat, 35; fibre, 50; carbohydrate, 600. After an adaptation period, the mice were separated into four groups (five mice per group) according to blood glucose levels and body weight. Mice were administered intraperitoneal (i.p.) injections of freshly prepared streptozotocin (STZ; 40 mg kg−1 in 0.01 mol L−1 citrate buffer) for 5 days, while mice in the normal control group were injected with the buffer only. Five days after injection, blood was collected from the tip of the tail vein, and the fasting glucose levels were measured.25 Blood glucose levels were monitored every week after a 12 h fast with venous blood from the tail vein using a glucose analyser (Superglucocard II; Arkray Inc., Tokyo, Japan) utilising the glucose oxidase method. The oral glucose tolerance test (OGTT) was performed during the last week of the experimental period. Mice that had fasted for 12 h received an oral load of 2 g glucose kg−1 of body weight. Blood glucose levels were measured before the load and at 30, 60, 90, 120 and 150 min after glucose administration.26
Oil-red O staining Cells were treated with silk peptide E5K6 in the adipogenic medium for days 0–8. For oil-red O staining of triglycerides,24 cells were washed gently with phosphate buffered saline twice, fixed with 10% neutral formalin for 1 h at room temperature and stained with 0.6% (w/v) oil-red O solution (60% isopropanol and 40% water) for at least 1 h. After staining of the lipid droplets, the oil-red O staining solution was removed and cells rinsed with water and dried. Images of the stained lipid droplets were collected on an Olympus microscope (Olympus, Tokyo, Japan). Subsequently, the dye retained in the cells was eluted with isopropanol and quantified by measuring the optical absorbance at 510 nm.
Statistical analysis All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 12.0 (SPSS Inc., Chicago, IL, USA). The differences among groups were evaluated by one-way analysis of variance (ANOVA) and Duncan’s multiple range tests. All data are reported as means ± standard deviations (SD).
Western blot analysis Washed cell pellets were lysed in cell lysis buffer (iNtRON Biotech, Seongnam, Korea). The protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL, USA). Proteins were separated on 10% sodium dodecyl sulfate–polyacrylamide gels and transferred onto nitrocellulose membranes. After 1 h of incubation in blocking solution (5% skim milk), the membranes were incubated for 1 h at a 1:1000 dilution of primary antibodies at room temperature. The membranes were washed three times with Tween-20/tris(hydroxymethyl)aminomethane-buffered saline (TTBS) and incubated in a 1:1000 dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The membranes were again washed three times with TTBS and then developed using ECL™ detection reagents. The autoradiograms were quantified by optical densitometry using the ImageJ program (NIH, Washington, DC, USA).
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Blood glucose and the oral glucose tolerance test The experimental protocol was reviewed and approved by the Korea University Animal Care Committee. Male ICR mice weighing 20 g (4 weeks old) were used in this study from Central Lab. Animal Inc. (Seoul, Korea). They were individually housed in plastic cages with grated stainless steel floors. The animal room was maintained at 24 ± 1 ∘ C with 60% atmospheric humidity, and a 12 h light/12 h dark cycle. The mice had ad libitum access to water and to a
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RESULTS Cytotoxicity of the tri-peptides GEY and GYG We use the MTT assay to examine the cytotoxicity of the synthetic tri-peptides in 3 T3-L1 cells differentiated into adipocytes. At a concentration of 1 mmol L−1 , GEY and GYG caused the death of 12% and 18% of the cells, respectively, compared with untreated controls (Fig. 2). Therefore, GEY and GYG concentrations lower than 500 μmol L−1 were used in all our studies.
Figure 2. Cytotoxicity of the tri-peptides GEY and GYG on 3 T3-L1 adipocytes.
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Figure 3. Effects of insulin and synthetic peptides on glucose uptake in 3 T3-L1 adipocytes. Differentiated 3 T3-L1 cells were incubated with synthetic peptides in the presence or absence of 0.2 nmol L−1 insulin for 15 min, and then assayed for glucose uptake activity. For measurement of glucose transport, 2-DG and glucose, at a final concentration of 3.7 kBq mL−1 (0.1 μCi mL−1 ) and 1 mmol L−1 , respectively, were added for 15 min at the end of incubation. The amount of 2-DG uptake was determined on a scintillation counter. The data are expressed as % of counts per minute (CPM) of sample/CPM of untreated controls. We used proglitazone as a positive control, and its glucose uptake activity was 139%. The glucose uptake activity of insulin 10 nmol L−1 was 190%. Data are means ± SD (n = 4) from three or more independent experiments. *P < 0.05 vs. absence of synthetic peptide; # P < 0.05 vs. absence of insulin.
of the synthetic peptides on glucose uptake with or without insulin in differentiated 3 T3-L1 adipocytes. In a previous study of 3 T3-L1 adipocytes, insulin-stimulated glucose uptake increased in a dose-dependent manner up to a concentration of 10 nmol L−1 .14 Glucose uptake at an insulin concentration of 10 nmol L−1 was 3.5-fold higher than in the absence of insulin treatment. Thus, treatment with insulin at 10 nmol L−1 was used as an index of maximum glucose uptake, and treatment with 0.2 nmol L−1 insulin was used to confirm the anti-diabetic activity of the synthetic peptides in 3 T3-L1 fibroblasts.27 Synthetic peptides induced a dose-dependent increase in glucose uptake rates in 3 T3-L1 adipocytes (Fig. 3). GEY and GYG at 250 μmol L−1 increased glucose uptake by 124% and 125% of control, respectively (P < 0.05) and the maximal effect was observed at 500 μmol L−1 , which increased glucose uptake by 128% and 136% of control, respectively (P < 0.05). The synthetic peptides caused a significant increase in glucose uptake in the presence of 0.2 nmol L−1 insulin, in comparison to control conditions (P < 0.05). Glucose transporter expression was also examined. GLUT4 expression was significantly increased my both tri-peptide, significantly (Fig. 4.)
Figure 4. Expression of GLUT1 and GLUT4 in 3 T3-L1 cells. 3 T3-L1 cells differentiated into adipocytes were washed once with low-glucose DMEM and incubated in the same medium for 2 h at 37 ∘ C for glucose starvation. The cells were incubated with KRH buffer along with the addition of the sample with or without 0.2 nmol L−1 insulin at 37 ∘ C for 2 h, and the cells were harvested for protein isolation. Protein levels were determined by densitometry and normalised to levels of GAPDH. Values are means ± SD (n = 3). Asterisk indicates significant differences among groups for each time point at P < 0.05 by Duncan’s multiple range test.
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Effect of the tri-peptides GEY and GYG on glucose uptake in 3T3-L1 adipocytes Insulin resistance is defined by a decrease of insulin sensitivity and glucose uptake in peripheral tissues, such as skeletal muscle and adipocyte tissue. Therefore, we examined the effects
Effect of the tri-peptides GEY and GYG on glucose tolerance The anti-hyperglycaemic effect of GYG was more evident in the OGTT (Fig. 5). Blood glucose levels at the beginning of the assay did not differ between the groups. After 150 min, mice in the diabetic control (DM control, STZ-induced mice untreated) group were unable to restore normal (control, ICR mice untreated) glucose levels. Meanwhile, diabetic mice treated with GYG exhibited decreased plasma glucose levels when compared to DM controls, although the difference was not significant. Glucose levels with a 50 mg kg−1 dose of GYG were lower than levels with a dose of 10 mg kg−1 (P < 0.05) 90 min after administration. Therefore, GYG treatment improved glucose tolerance in a dose-dependent manner for the duration of the OGTT in STZ-induced diabetic mice.
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Figure 5. Glucose tolerance test in the presence of the synthetic peptide GYG. The glucose level (A) as well as corresponding area under the curve (AUC) is presented (n = 6). (B) Calculated area under the curve. a–d Different letters indicate significant difference at P < 0.05.
DISCUSSION
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In the present study, we observed that synthetic peptides stimulated basal and insulin-mediated glucose uptake in insulin-sensitive cells, although the potency of these synthetic peptides was much lower than that of insulin (Fig. 3). Previous studies from our laboratory demonstrated that the silk peptide E5K6 reduces blood glucose level.13,14 Because glucose uptake in insulin target tissues is a critical step in maintaining glucose homeostasis and in clearing the postprandial glucose load,18 our results may partially explain the mechanism mediating the hypoglycaemic effects of E5K6. To understand the mechanism whereby synthetic peptides stimulate glucose transport, we examined the effects of these compounds on GLUT1 and GLUT4 cellular localisation and on the insulin signalling pathway in 3 T3-L1 fibroblasts induced to differentiate into adipocytes. GLUT1 and GLUT4 are essential mediators of glucose uptake in adipocytes.18,28 Moreover, our findings demonstrate that synthetic peptides stimulated basal and insulin-mediated glucose uptake in adipocytes and simultaneously promoted the translocation of GLUT1 and GLUT4. Similarly, Kim et al.29 reported that a synthetic hexapeptide, Gly-Ala-Gly-Val-Gly-Tyr, increases both basal and insulin-stimulated glucose uptake through induction of GLUT1 expression and GLUT4 translocation mediated by PI3K. Hyun et al.11 reported that silk protein hydrolysate increased GLUT1 and GLUT4 by 1.5- and 2.7-fold, respectively. Moreover, GYG treatment improved glucose tolerance over the entire OGTT in STZ-induced diabetic mice in a dose-dependent manner (Fig. 5). Insulin-mediated glucose transport to appropriate tissues is essential for maintenance of glucose homeostasis.18 Adipocytes and muscle cells are cells highly sensitive to insulin. In adipocytes, insulin stimulates glucose transport by enhancing the translocation of GLUT4 and, to a lesser extent, of GLUT1 from the cytosol to the plasma membrane (PM).19,20 The classical pathway of insulin signalling, involving glucose transporter translocation to the cell surface, is induced by autophosphorylation of the insulin receptor on multiple tyrosine residues following insulin binding. These events result in the tyrosine phosphorylation of a family of insulin receptor substrate proteins and activation of a complex network of downstream signal kinases, such as phosphatidylinositol 3-kinase and the serine/threonine kinase protein kinase B.19,21
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In previous work, we reported that E5K6 simultaneously stimulates glucose uptake and suppresses fat accumulation; however, a crude preparation was used in this studies, and the active components remain undetermined. We identified two tri-peptides, GEY and GYG, that inhibit 𝛼-glucosidase activity. In the present study, we evaluated the stimulating effect of the GEY and GYG tri-peptides on glucose uptake. Our results demonstrate that the GEY and GYG tri-peptides stimulate glucose transport in adipocytes by inducing the expression of GLUT1 and GLUT4, mediated by activation of insulin-like signalling pathways. Administration of silk peptides, such as mixtures of hydrolysed silk, or those derived from silk, including GYG and GEY, could be a new strategy for the development of anti-diabetic nutraceutical peptides. We hypothesised that GYG, a tri-peptide derived from E5K6, would enhance insulin-stimulated glucose uptake through up-regulation of GLUT and decreased fat accumulation. Increased glucose uptake via up-regulation of GLUT 4 and decreased fat accumulation mediated by GYG confirmed our hypothesis. In conclusion, E5K6 is a promising source of anti-diabetic peptides and the cocoon, a by-product of the sericulture, might be utilised for this novel application.
REFERENCES 1 Davis SN and Granner DK, Insulin, oral hypoglycemic agents, and the pharmacology of the endocrine pancreas, in Goodman & Gilman’s, The Pharmacological Basis of Therapeutics, 10th edition. McGrow Hill, New York, pp. 1704–1705 (1996). 2 Yeh GY, Eisenberg DM, Kaptchuk TJ and Phillips RS, Systematic review of herbs and dietary supplements for glycemic control in diabetes. Diabetes Care 26:1277–1294 (2003). 3 Pari L and Umamaheswari J, Antihyperglycaemic activity of Musa sapientum flowers: effect on lipid peroxidation in alloxan diabetic rats. Phytother Res 14:136–138 (2000). 4 Tiwari AK and Rao JM, Diabetes mellitus and multiple therapeutic approaches of phytochemicals: Present status and future prospects. Curr Sci 83:30–38 (2002). 5 Kurosaki S, Otsuka H, Kunitomo M, Koyama M, Pawankar R and Matumoto K, Fibroin allergy: IgE mediated hypersensitivity to silk suture materials. Nippon Ika Daigaku Zasshi 66:41–44 (1999). 6 Santin M, Motta A, Freddi G and Cannas M, In vitro evaluation of the inflammatory potential of the silk fibroin. J Biomed Mater Res 46:382–389 (1999). 7 Gotoh K, Izumi H, Kanamoto T, Tamada Y and Nakashima H, Sulfated fibroin, a novel sulfated peptide derived from silk, inhibits
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8 9 10 11 12
13 14 15 16 17 18 19
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human immunodeficiency virus replication in vitro. Biosci Biotechnol Biochem 64:1664–1670 (2000). Shin S, Yeon S, Park D, Oh J, Kang H, Kim S, et al, Silk amino acids improve physical stamina and male reproductive function of mice. Biol Pharm Bull 33:273–278 (2010). Singh TM, Kadowaki MH, Glagov S and Zarins CK, Role of fibrinopeptide B in early atherosclerotic lesion formation. Am J Surg 160:156–159 (1990). Gao J, Bai J, Man Q and Liu G, Effects of silk hydrates on the blood glucose metabolism in rats with experimental diabetes. J Hygiene Res 29:223–225 (2000). Hyun CK, Kim IY and Frost SC, Soluble fibroin enhances insulin sensitivity and glucose metabolism in 3 T3-L1 adipocytes. J Nutr 134:3257–3263 (2004). Lee YS, Park MJ, Choi JE, Kim JY, Nam MS and Jeong YH, Effects of silk protein hydrolysates on blood glucose level, serum insulin and leptin secretion in OLETF rats. J Korean Soc Food Sci Nutr 36:703–707 (2007). Jung EY, Lee HS, Lee HJ, Kim JM, Lee KW and Suh HJ, Feeding silk protein hydrolysates to C57BL/KsJ-db/db mice improves blood glucose and lipid profiles. Nutr Res 30:783–790 (2010). Lee HS, Lee HJ and Suh HJ, Silk protein hydrolysate increases glucose uptake through up-regulation of GLUT 4 and reduces the expression of leptin in 3 T3-L1 fibroblast. Nutr Res 11:35–41 (2011). Lee HJ, Lee HS, Choi JW, Ra KS, Kim JM and Suh HJ, Novel tripeptides with 𝛼-glucosidase inhibitory activity isolated from silk cocoon hydrolysate. J Agric Food Chem 59:11522–11525 (2011). Zhou CZ, Confalonieri F, Jacquet M, Perasso R, Li CH and Janin J, Silk fibroin: Structural implications of a remarkable amino acid sequence. Proteins Struct Funct Genet 44:119–122 (2001). Zhou CZ, Confalonieri F, Medina N, Zivanovic Y, Esnault C, Yang T, et al, Fine organization of Bombyx mori fibroin heavy chain gene. Nucleic Acid Res 28:2413–2419 (2000). Herman MA and Kahn BB, Glucose transport and sensing in the maintenance of glucose homeostasis and metabolic harmony. J Clin Invest 116:1767 (2006). Watson RT, Kanzaki M and Pessin JE, Regulated membrane trafficking of the insulin-responsive glucose transporter 4 in adipocytes. Endocrine Rev 25:177–204 (2004).
20 Weiland M, Schürmann A, Schmidt W and Joost H, Development of the hormone-sensitive glucose transport activity in differentiating 3 T3-L1 murine fibroblasts. Role of the two transporter species and their subcellular localization. Biochem J 270:331 (1990). 21 Khan A and Pessin J, Insulin regulation of glucose uptake: A complex interplay of intracellular signalling pathways. Diabetologia 45:1475–1483 (2002). 22 Liu F, Kim J, Li Y, Liu X, Li J and Chen X, An extract of Lagerstroemia speciosa L. has insulin-like glucose uptake–stimulatory and adipocyte differentiation–inhibitory activities in 3 T3-L1 cells. J Nutr 131:2242–2247 (2001). 23 Roffey B, Atwal AS, Johns T and Kubow S, Water extracts from Momordica charantia increase glucose uptake and adiponectin secretion in 3 T3-L1 adipose cells. J Ethnopharmacol 112:77 (2007). 24 Havel PJ, Role of Adipose Tissue in Body-weight Regulation: Mechanisms Regulating Leptin Production and Energy Balance. Cambridge University Press, Cambridge, pp. 359–371 (2000). 25 Takamura T, Ando H, Nagai Y, Yamashita H, Nohara E and Kobayashi K, Pioglitazone prevents mice from multiple low-dose streptozotocin-induced insulitis and diabetes. Diabetes Res Clin Pract 44:107–114 (1999). 26 Han GC, Ko SK, Sung JH and Chung SH, Compound K enhances insulin secretion with beneficial metabolic effects in db/db mice. J Agric Food Chem 55:10641–10648 (2007). 27 Ko BS, Choi SB, Park SK, Jang JS, Kim YE and Park S, Insulin sensitizing and insulinotropic action of berberine from Cortidis rhizoma. Biol Pharm Bull 28:1431–1437 (2005). 28 Liao W, Nguyen MTA, Imamura T, Singer O, Verma IM and Olefsky JM, Lentiviral short hairpin ribonucleic acid-mediated knockdown of GLUT4 in 3 T3-L1 adipocytes. Endocrinology 147:2245–2252 (2006). 29 Kim ED, Kim E, Lee JH and Hyun CK, Gly-Ala-Gly-Val-Gly-Tyr, a novel synthetic peptide, improves glucose transport and exerts beneficial lipid metabolic effects in 3 T3-L1 adipoctyes. Eur J Pharmacol 650:479–485 (2011).
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J Sci Food Agric 2016; 96: 116–121
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/jsfa
Revised: 12 December 2014
Accepted article published: 31 December 2014
Published online in Wiley Online Library: 2 February 2015
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7067
Hypoglycaemic effects of functional tri-peptides from silk in differentiated adipocytes and streptozotocin-induced diabetic mice Bok Kyung Han,a† Hyun Jung Lee,b† Hyun-Sun Lee,c Hyung Joo Suhb and Yooheon Parkb* Abstract BACKGROUND: In this study, the tri-peptides Gly-Glu-Tyr (GEY) and Gly-Tyr-Gly (GYG), identified previously as active compounds from the silk peptide E5K6, significantly stimulated basal and insulin-mediated glucose uptake by 3T3-L1 fibroblasts in a dose-dependent manner. RESULTS: Synthetic GEY and GYG peptides at a concentration of 500 𝛍mol L−1 significantly increased glucose transporter type 4 expression by 157% and 239%, respectively. Differentiation of 3T3-L1 cells into adipocytes leads to accumulation of intracellular fat droplets, and GEY and GYG at a concentration of 250 𝛍mol L−1 suppressed this effect by 72% and 75%, respectively. GYG improved glucose tolerance in steptozotocin (STZ)-induced diabetic mice in a dose-dependent manner. CONCLUSION: These results suggest that GYG isolated from E5K6 has anti-diabetic potential and silk waste products containing bioactive peptides could be used to the developments of treatments to lower blood glucose. © 2014 Society of Chemical Industry Keywords: silk peptide E5K6; 3T3-L1 adipocyte; glucose uptake; STZ-induced diabetic mice; alcalase
INTRODUCTION
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Diabetes mellitus (DM) is a major endocrine disorder and a global public health concern, affecting nearly 10% of the world’s population. DM comprises a group of syndromes characterised by hyperglycaemia, altered metabolism of lipids, carbohydrates and proteins, and an increased risk of complications from vascular disease.1 Conventional drugs used for DM treatment present significant limitations such as strict and multiple dosing regimens, high cost, inaccessibility, and adverse effects. Studies of alternative treatments for DM have provided valuable leads for the development of therapeutic strategies. In the last few decades, there has been exponential growth in the field of alternative treatments by natural supplements, owing to their natural origin and diminished side effects.2 Because natural products might be less toxic and present fewer side effects than synthetic compounds,3 they might have therapeutic potential for complex disorders, such as diabetes and associated complications.4 Silk is a type of natural fibre produced by silkworms (Bombyx mori). Since the 1970s, many bioactive peptides derived from food protein hydrolysates have been studied as potential nutraceuticals. Based on this body of work, application of silk peptides in the biotechnological and biomedical fields has received increasing attention.5,6 Silk peptides have been employed as a biomedical suture material and are believed to be safe for humans. Silk J Sci Food Agric 2016; 96: 116–121
peptides consist of peptides of different sizes, which create peptides that are 18–20 amino acids in length and enzymatic degradation results in peptides of specific sizes or compositions presenting diverse bioactivities, such as hypocholesterolaemic, antioxidant, immunoregulatory, anti-tumour, antiviral, and antibacterial effects.7 – 9 Previous studies have reported that silk peptides influence blood glucose, insulin and serum lipids levels in vivo,10 – 12 but the underlying mechanisms mediating these effects were not clear because the active compound was not identified. Previous reports from our laboratory demonstrated that the silk peptide E5K6 reduces plasma glucose levels and improves the lipid profile in C57BL/KsJ-db/db mice, increases glucose uptake via up-regulation
∗
Correspondence to: Yooheon Park, Department of Food and Nutrition, Korea University, Seoul 136–703, Republic of Korea. E-mail: [email protected]
† Bok Kyung Han and Hyun Jung Lee contributed equally to this study. a BK Bio Co. Ltd, Sungnam, 462-819, Republic of Korea b Department of Food and Nutrition, Korea University, Seoul 136-703, Republic of Korea c Food Quality & Safety Department, Agency for Korea National Food Cluster, Gwacheon, 427-806, Republic of Korea
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Hypoglycaemic effects of peptides from silk
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Figure 1. Chemical structure of the tri-peptides GEY and GYG (After a previously published figure15 ).
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MATERIALS AND METHODS Materials The synthetic peptides GEY and GYG were obtained from Peptron (Daejeon, Korea). 3 T3-L1 fibroblast cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cell culture reagents such as low-glucose Dulbecco’s modified Eagle’s medium (DMEM), high-glucose DMEM, fetal bovine serum (FBS), and antibiotics (100 U mL−1 penicillin and 100 μg mL−1 streptomycin) were purchased from Gibco BRL (Grand Island, NY, USA). 2-Deoxy-D-[1,2-3 H] glucose (2-DG) was purchased from PerkinElmer Life (Boston, MA, USA). 3-Isobutylmethyl xanthine (IBMX), dexamethasone, insulin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St Louis, MO, USA). General chemicals and laboratory equipment were purchased from standard sources.
Conditions of cell culture 3 T3-L1 cells were cultured and induced to differentiate into adipocytes as described previously.22 Pre-adipocytes were cultured in high-glucose DMEM with 10% FBS and 1% penicillin–streptomycin at 37 ∘ C in a humidified atmosphere of 5% CO2 , and cells from passages 3 to 20 were used. Cells plated onto six- or 24-well plates were grown to confluence. Two days after reaching confluence (day 0), cells were induced to differentiate in high-glucose DMEM supplemented with 10% FBS, 1 μg mL−1 insulin, 50 mmol L−1 dexamethasone, and 0.8 mmol L−1 IBMX. Two days following the initiation of cellular differentiation (day 2), dexamethasone and IBMX were withdrawn from the media, whereas insulin was withdrawn from the media 4 days after initiation of differentiation (day 4). The differentiated adipocytes were maintained in high-glucose DMEM and 10% FBS until maturity (days 9–12). On the day of the experiment, each well was examined under a microscope to identify the percentage of undifferentiated cells not resembling adipocytes, which are round cells rich in easily distinguishable fat globules. The wells that were used for experimentation contained little to no pre-adipocyte cells and were easily distinguishable as >90% differentiated overall.
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of glucose transporter type (GLUT) 4 and decreases fat accumulation by up-regulating leptin, thereby exhibiting anti-diabetic effects.13,14 Subsequently, by means of 𝛼-glucosidase inhibition assays and amino acid sequencing, we identified and purified two tri-peptides, Gly-Glu-Tyr (GEY) and Gly-Tyr-Gly (GYG), from E5K6. The structure was elucidated, and we reported that these compounds have an anti-diabetic effect (Fig. 1).15 The composition of fibroin protein is 45.9% Gly, 30.3% Ala, 12.1% Ser, 5.3% Tyr, 1.8% Val and 4.7% of the other 15 amino acids. Most of the sequence exhibits low-complexity and 94% of the sequence is occupied by Gly-X dipeptide motifs, where the probability of X being Ala, Ser and Tyr is 64%, 22% and 10%, respectively.16,17 Insulin-mediated glucose transport to appropriate tissues is essential for maintenance of glucose homeostasis.18 Adipocytes and muscle cells are cells highly sensitive to insulin. In adipocytes, insulin stimulates glucose transport by enhancing the translocation of GLUT4 and, to a lesser extent, of GLUT1 from the cytosol to the plasma membrane.19,20 The classical pathway of insulin signalling, involving glucose transporter translocation to the cell surface, is induced by autophosphorylation of the insulin receptor on multiple tyrosine residues following insulin binding. These events result in the tyrosine phosphorylation of a family of insulin receptor substrate (IRS) proteins and activation of a complex network of downstream signal kinases, such as phosphatidylinositol 3-kinase and the serine/threonine kinase protein kinase B.19,21 In previous work, we reported that E5K6 simultaneously stimulates glucose uptake and suppresses fat accumulation; however, a crude preparation was used in this studies, and the active components remain undetermined. We identified two tri-peptides, GEY and GYG, that inhibit 𝛼-glucosidase activity. In the present study, we evaluated the stimulating effect of the GEY and GYG tri-peptides on glucose uptake and their inhibitory effects on fat accumulation in 3 T3-L1 fibroblasts. Our results demonstrate that the GEY and GYG tri-peptides stimulate glucose transport in adipocytes by inducing the translocation of GLUT1 and GLUT4, mediated by activation of insulin-like signalling pathways. This research clarifies the effects of the anti-hyperglycaemic and anti-hyperlipidaemic activities of silk protein hydrolysates.
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Insulin-stimulated glucose uptake assay in 3T3-L1 adipocyte cells Glucose uptake activity was determined in six-well plates. The effects of silk peptide on insulin-stimulated glucose uptake were determined in 3 T3-L1 fibroblasts induced to differentiate into adipocytes for 9–12 days in high glucose DMEM. Glucose uptake activity was analysed by measuring the uptake of 2-DG.23 Differentiated 3 T3-L1 cells were glucose starved following two washes with low glucose DMEM and incubation with 2 mL of low glucose DMEM containing 10% FBS, at 37 ∘ C for 2 h. After washing with Krebs–Ringer–Hepes (KRH) buffer (128 mmol L−1 NaCl, 4.7 mmol L−1 KCl, 1.25 mmol L−1 CaCl2 , 1.25 mmol L−1 MgSO4 , 10.0 mmol L−1 Na2 HPO4 , 20 mmol L−1 Hepes), the cells were incubated with KRH buffer in the presence of sample, with or without 0.2 nmol L−1 insulin at 37 ∘ C for 30 min. Subsequently, 2-DG and glucose, at a final concentration of 3.7 kBq mL−1 (0.1 μCi mL−1 ) and 1 mmol L−1 , respectively, were added for 15 min. Following addition of cold phosphate buffered saline, the cells were digested with 1% Triton X-100 for 40 min at 37 ∘ C and counted by a scintillation counter (Beckman LS 6000; Beckman, Pasadena, CA, USA).
commercial diet (Samyang, Gumi, Korea) containing the following (g 100 kg−1 of diet): moisture, 80; protein, 230; fat, 35; fibre, 50; carbohydrate, 600. After an adaptation period, the mice were separated into four groups (five mice per group) according to blood glucose levels and body weight. Mice were administered intraperitoneal (i.p.) injections of freshly prepared streptozotocin (STZ; 40 mg kg−1 in 0.01 mol L−1 citrate buffer) for 5 days, while mice in the normal control group were injected with the buffer only. Five days after injection, blood was collected from the tip of the tail vein, and the fasting glucose levels were measured.25 Blood glucose levels were monitored every week after a 12 h fast with venous blood from the tail vein using a glucose analyser (Superglucocard II; Arkray Inc., Tokyo, Japan) utilising the glucose oxidase method. The oral glucose tolerance test (OGTT) was performed during the last week of the experimental period. Mice that had fasted for 12 h received an oral load of 2 g glucose kg−1 of body weight. Blood glucose levels were measured before the load and at 30, 60, 90, 120 and 150 min after glucose administration.26
Oil-red O staining Cells were treated with silk peptide E5K6 in the adipogenic medium for days 0–8. For oil-red O staining of triglycerides,24 cells were washed gently with phosphate buffered saline twice, fixed with 10% neutral formalin for 1 h at room temperature and stained with 0.6% (w/v) oil-red O solution (60% isopropanol and 40% water) for at least 1 h. After staining of the lipid droplets, the oil-red O staining solution was removed and cells rinsed with water and dried. Images of the stained lipid droplets were collected on an Olympus microscope (Olympus, Tokyo, Japan). Subsequently, the dye retained in the cells was eluted with isopropanol and quantified by measuring the optical absorbance at 510 nm.
Statistical analysis All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 12.0 (SPSS Inc., Chicago, IL, USA). The differences among groups were evaluated by one-way analysis of variance (ANOVA) and Duncan’s multiple range tests. All data are reported as means ± standard deviations (SD).
Western blot analysis Washed cell pellets were lysed in cell lysis buffer (iNtRON Biotech, Seongnam, Korea). The protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL, USA). Proteins were separated on 10% sodium dodecyl sulfate–polyacrylamide gels and transferred onto nitrocellulose membranes. After 1 h of incubation in blocking solution (5% skim milk), the membranes were incubated for 1 h at a 1:1000 dilution of primary antibodies at room temperature. The membranes were washed three times with Tween-20/tris(hydroxymethyl)aminomethane-buffered saline (TTBS) and incubated in a 1:1000 dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The membranes were again washed three times with TTBS and then developed using ECL™ detection reagents. The autoradiograms were quantified by optical densitometry using the ImageJ program (NIH, Washington, DC, USA).
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Blood glucose and the oral glucose tolerance test The experimental protocol was reviewed and approved by the Korea University Animal Care Committee. Male ICR mice weighing 20 g (4 weeks old) were used in this study from Central Lab. Animal Inc. (Seoul, Korea). They were individually housed in plastic cages with grated stainless steel floors. The animal room was maintained at 24 ± 1 ∘ C with 60% atmospheric humidity, and a 12 h light/12 h dark cycle. The mice had ad libitum access to water and to a
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RESULTS Cytotoxicity of the tri-peptides GEY and GYG We use the MTT assay to examine the cytotoxicity of the synthetic tri-peptides in 3 T3-L1 cells differentiated into adipocytes. At a concentration of 1 mmol L−1 , GEY and GYG caused the death of 12% and 18% of the cells, respectively, compared with untreated controls (Fig. 2). Therefore, GEY and GYG concentrations lower than 500 μmol L−1 were used in all our studies.
Figure 2. Cytotoxicity of the tri-peptides GEY and GYG on 3 T3-L1 adipocytes.
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Figure 3. Effects of insulin and synthetic peptides on glucose uptake in 3 T3-L1 adipocytes. Differentiated 3 T3-L1 cells were incubated with synthetic peptides in the presence or absence of 0.2 nmol L−1 insulin for 15 min, and then assayed for glucose uptake activity. For measurement of glucose transport, 2-DG and glucose, at a final concentration of 3.7 kBq mL−1 (0.1 μCi mL−1 ) and 1 mmol L−1 , respectively, were added for 15 min at the end of incubation. The amount of 2-DG uptake was determined on a scintillation counter. The data are expressed as % of counts per minute (CPM) of sample/CPM of untreated controls. We used proglitazone as a positive control, and its glucose uptake activity was 139%. The glucose uptake activity of insulin 10 nmol L−1 was 190%. Data are means ± SD (n = 4) from three or more independent experiments. *P < 0.05 vs. absence of synthetic peptide; # P < 0.05 vs. absence of insulin.
of the synthetic peptides on glucose uptake with or without insulin in differentiated 3 T3-L1 adipocytes. In a previous study of 3 T3-L1 adipocytes, insulin-stimulated glucose uptake increased in a dose-dependent manner up to a concentration of 10 nmol L−1 .14 Glucose uptake at an insulin concentration of 10 nmol L−1 was 3.5-fold higher than in the absence of insulin treatment. Thus, treatment with insulin at 10 nmol L−1 was used as an index of maximum glucose uptake, and treatment with 0.2 nmol L−1 insulin was used to confirm the anti-diabetic activity of the synthetic peptides in 3 T3-L1 fibroblasts.27 Synthetic peptides induced a dose-dependent increase in glucose uptake rates in 3 T3-L1 adipocytes (Fig. 3). GEY and GYG at 250 μmol L−1 increased glucose uptake by 124% and 125% of control, respectively (P < 0.05) and the maximal effect was observed at 500 μmol L−1 , which increased glucose uptake by 128% and 136% of control, respectively (P < 0.05). The synthetic peptides caused a significant increase in glucose uptake in the presence of 0.2 nmol L−1 insulin, in comparison to control conditions (P < 0.05). Glucose transporter expression was also examined. GLUT4 expression was significantly increased my both tri-peptide, significantly (Fig. 4.)
Figure 4. Expression of GLUT1 and GLUT4 in 3 T3-L1 cells. 3 T3-L1 cells differentiated into adipocytes were washed once with low-glucose DMEM and incubated in the same medium for 2 h at 37 ∘ C for glucose starvation. The cells were incubated with KRH buffer along with the addition of the sample with or without 0.2 nmol L−1 insulin at 37 ∘ C for 2 h, and the cells were harvested for protein isolation. Protein levels were determined by densitometry and normalised to levels of GAPDH. Values are means ± SD (n = 3). Asterisk indicates significant differences among groups for each time point at P < 0.05 by Duncan’s multiple range test.
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Effect of the tri-peptides GEY and GYG on glucose uptake in 3T3-L1 adipocytes Insulin resistance is defined by a decrease of insulin sensitivity and glucose uptake in peripheral tissues, such as skeletal muscle and adipocyte tissue. Therefore, we examined the effects
Effect of the tri-peptides GEY and GYG on glucose tolerance The anti-hyperglycaemic effect of GYG was more evident in the OGTT (Fig. 5). Blood glucose levels at the beginning of the assay did not differ between the groups. After 150 min, mice in the diabetic control (DM control, STZ-induced mice untreated) group were unable to restore normal (control, ICR mice untreated) glucose levels. Meanwhile, diabetic mice treated with GYG exhibited decreased plasma glucose levels when compared to DM controls, although the difference was not significant. Glucose levels with a 50 mg kg−1 dose of GYG were lower than levels with a dose of 10 mg kg−1 (P < 0.05) 90 min after administration. Therefore, GYG treatment improved glucose tolerance in a dose-dependent manner for the duration of the OGTT in STZ-induced diabetic mice.
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Figure 5. Glucose tolerance test in the presence of the synthetic peptide GYG. The glucose level (A) as well as corresponding area under the curve (AUC) is presented (n = 6). (B) Calculated area under the curve. a–d Different letters indicate significant difference at P < 0.05.
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In the present study, we observed that synthetic peptides stimulated basal and insulin-mediated glucose uptake in insulin-sensitive cells, although the potency of these synthetic peptides was much lower than that of insulin (Fig. 3). Previous studies from our laboratory demonstrated that the silk peptide E5K6 reduces blood glucose level.13,14 Because glucose uptake in insulin target tissues is a critical step in maintaining glucose homeostasis and in clearing the postprandial glucose load,18 our results may partially explain the mechanism mediating the hypoglycaemic effects of E5K6. To understand the mechanism whereby synthetic peptides stimulate glucose transport, we examined the effects of these compounds on GLUT1 and GLUT4 cellular localisation and on the insulin signalling pathway in 3 T3-L1 fibroblasts induced to differentiate into adipocytes. GLUT1 and GLUT4 are essential mediators of glucose uptake in adipocytes.18,28 Moreover, our findings demonstrate that synthetic peptides stimulated basal and insulin-mediated glucose uptake in adipocytes and simultaneously promoted the translocation of GLUT1 and GLUT4. Similarly, Kim et al.29 reported that a synthetic hexapeptide, Gly-Ala-Gly-Val-Gly-Tyr, increases both basal and insulin-stimulated glucose uptake through induction of GLUT1 expression and GLUT4 translocation mediated by PI3K. Hyun et al.11 reported that silk protein hydrolysate increased GLUT1 and GLUT4 by 1.5- and 2.7-fold, respectively. Moreover, GYG treatment improved glucose tolerance over the entire OGTT in STZ-induced diabetic mice in a dose-dependent manner (Fig. 5). Insulin-mediated glucose transport to appropriate tissues is essential for maintenance of glucose homeostasis.18 Adipocytes and muscle cells are cells highly sensitive to insulin. In adipocytes, insulin stimulates glucose transport by enhancing the translocation of GLUT4 and, to a lesser extent, of GLUT1 from the cytosol to the plasma membrane (PM).19,20 The classical pathway of insulin signalling, involving glucose transporter translocation to the cell surface, is induced by autophosphorylation of the insulin receptor on multiple tyrosine residues following insulin binding. These events result in the tyrosine phosphorylation of a family of insulin receptor substrate proteins and activation of a complex network of downstream signal kinases, such as phosphatidylinositol 3-kinase and the serine/threonine kinase protein kinase B.19,21
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In previous work, we reported that E5K6 simultaneously stimulates glucose uptake and suppresses fat accumulation; however, a crude preparation was used in this studies, and the active components remain undetermined. We identified two tri-peptides, GEY and GYG, that inhibit 𝛼-glucosidase activity. In the present study, we evaluated the stimulating effect of the GEY and GYG tri-peptides on glucose uptake. Our results demonstrate that the GEY and GYG tri-peptides stimulate glucose transport in adipocytes by inducing the expression of GLUT1 and GLUT4, mediated by activation of insulin-like signalling pathways. Administration of silk peptides, such as mixtures of hydrolysed silk, or those derived from silk, including GYG and GEY, could be a new strategy for the development of anti-diabetic nutraceutical peptides. We hypothesised that GYG, a tri-peptide derived from E5K6, would enhance insulin-stimulated glucose uptake through up-regulation of GLUT and decreased fat accumulation. Increased glucose uptake via up-regulation of GLUT 4 and decreased fat accumulation mediated by GYG confirmed our hypothesis. In conclusion, E5K6 is a promising source of anti-diabetic peptides and the cocoon, a by-product of the sericulture, might be utilised for this novel application.
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