Compound K, a final intestinal metabolite of ginsenosides, enhances insulin secretion in MIN6 pancreatic β-cells by upregulation of GLUT2

Журнал(книга):

Раздел науки:

Читать онлайн: 
Текст статьи: 
Jian Gu a,1, Wei Li a,b,1, Dong Xiao a, ShengNan Wei a, WanLi Cui a, WeiJia Chen a, YaLi Hu a, XiaoJia Bi a, YongChol Kim d, Jing Li a, HongWei Du c,⁎, Ming Zhang a,⁎⁎, Li Chen a a Department of Pharmacology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, China b College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun 1300118, China c Department of Pediatric Endocrinology, The First Clinical Hospital Affiliated to Jilin University, 130021, China d Department of Pharmacology, Basic Medical Faculty, Pyongyang Medical University, Kim IL Sung University, Democratic People's Republic of Korea a r t i c l e i n f o a b s t r a c t Article history: Received 25 January 2013 Accepted in revised form 19 March 2013 Available online 1 April 2013 Compound K (CK) is a final intestinal metabolite of protopanaxadiol-type ginsenosides from Panax ginseng and shows various bioactivities. Although it has also been found to have the property of anti-diabetes, the long-term effect of CK on insulin secretion in β-cells is still unclear. In this study, CK was prepared from ginsenoside Rd by snailase hydrolysis and its effect on the insulin secretion activity in MIN6 pancreatic β-cell lines in vitro was assessed. The expression of glucose transporter isoform-2 (GLUT2) and the cellular ATP content were also examined by western blot and HPLC analysis, respectively. The results showed that CK significantly enhanced insulin secretion, increased cellular ATP content, and upregulated the expression of GLUT2. These findings indicate that CK exerts prominent stimulatory effects on insulin secretion in the MIN6 cells partly via upregulating the expression of GLUT2. © 2013 Elsevier B.V. All rights reserved. Keywords: Compound K Insulin secretion MIN6 cells GLUT2 1. Introduction Ginsenoside compound K (CK) is a final metabolite of protopanaxadiol ginsenosides [1,2], which is also known as ginsenoside M1, or IH901. Recently, CK has received much attention due to its various bioactivities including anti-cancer [3,4], anti-inflammation [5], and hepatoprotective effect [6,7]. Moreover, CK has also been found to have the property of anti-diabetes. For example, previous studies showed that CK reduced hyperglycemia in db/db mice by stimulating insulin secretion and this action was presumably associated with an ATP-sensitive K+ channel. In addition, insulin secretion increased in HIT-T15 cells with one-hour CK incubation [8] and the effect of co-treatment with CK and metformin is much better than CK and metformin alone for the diabetic db/db mice [9], and our previous study has also confirmed hypolyglycemic effect of CK on type 2 diabetes mice induced by high-fat diet combining with streptozotocin (HFD/STZ) [10]. However, the effect on insulin secretion by long-term CK treatment in vitro and its possible mechanism still need further investigation. Insulin secretary function plays an important role in the development of diabetes. The change of blood glucose levels is a signal for pancreatic cells to release insulin [11]. Among the process of insulin secretion, the glucose transporter isoform-2 (GLUT2) is an important glucose sensor, which is a facilitative glucose transporter in the liver, pancreas, kidney, intestine, and brain [12]. Under physiological conditions, GLUT2 of pancreatic β-cells permits rapid glucose uptake into the cells and another glucose sensor-glucokinase controls the rate-limiting step for further glucose metabolism [13]. Lately, accumulated evidence suggests that GLUT2 may serve as a potential drug target for obesity and diabetes [14]. Therefore, the purpose of this study was to evaluate the chronic effect of CK on insulin secretion in Fitoterapia 87 (2013) 84–88 ⁎ Corresponding author. Tel.: +86 431 88782459. ⁎⁎ Corresponding author. Tel.: +86 431 85619799. E-mail addresses: shared-inbox@163.com (H. Du), chen_lab@163.com (M. Zhang). 1 These authors contributed equally to this work. 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.03.020 Contents lists available at SciVerse ScienceDirect Fitoterapia journal homepage: www.elsevier.com/locate/fitote vitro, and whether the GLUT2 was involved in the hypoglycemic effect of ginsenoside CK. 2. Materials and methods 2.1. Preparation of ginsenoside CK Ginsenoside Rd was isolated in our lab by the previous method [15]. The snailase was used to convert Rd to CK under optimized conditions. In brief, the snailase was incubated with Rd in a pH 4.5 sodium acetate buffer with agitation at a temperature of 50 °C and enzyme load of 20% for a reaction time of 24 h. The mixture was subsequently placed in a water bath at 90 °C to terminate the enzymatic reaction. The reaction mixtures were individually evaporated, dissolved in methanol, and separated through repeated silica column chromatography to obtain CK and the yield was 13.89 mg/mL. CK's purity was more than 98.0% by HPLC analysis. A HPLC method was developed using a reversed-phase C18 column (Hypersil ODS2, 250 mm × 4.6 mm I.D., 5 μm). The column temperature was set at room temperature and detection wavelength was set at 203 nm. The mobile phase was consisted of 25% acetonitrile with a flow rate of 1.0 mL/min. The 20 μL of sample solution was directly injected into the chromatographic column manually. 2.2. Cell culture MIN6 cell line was purchased from Xiangya Medical College of Central South University. The cells were grown in Dulbecco's modified Eagle's medium (DMEM 25 mmol/L glucose, GIBCO) equilibrated with 5% CO2 and 95% air at 37 °C. The medium was supplemented with 10% fetal calf serum, 100 U/mL penicillin sulfate and 50 μg/mL gentamicin. All experiments were performed when the cells reach 80% confluence. 2.3. MTT assay MIN6 cells were seeded in 96 well plates at 1 × 104 cells/ well, and treated in the absence or presence of CK (2, 4, 8, 16, and 32 μM) for 24 h at 37 °C. Cell viability was assessed using the MTT assay (Sigma Chemicals Co.) in accordance with the manufacturer's protocol. 2.4. Insulin secretion and content assays For insulin secretion experiments, MIN6 cells were seeded in 6-well plates. After 24 h incubation with CK-8 μM, the cells were preincubated at 37 °C for 0.5 h in Kreb-Ringer bicarbonate Hepes buffer (KRB; 129 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 5 mM NaHCO3, 10 mM Hepes, and 0.2% BSA). Cells were then treated in Kreb-Ringer bicarbonate Hepes buffer containing 3 mM glucose or 30 mM glucose for 1 h. Cell supernatants were collected and centrifuged at 1000 rpm, 4 °C for 5 min. Insulin was measured by radioimmunoassay (RIA) (Tianjin Nine Tripods Medical & Bioengineering Co., Ltd, Tianjin, China). Cells were lysed in lysis buffer: (1% Triton-X, 20 mM HEPES, pH 7.4, 100 mM KCl, 2 mM EDTA, 1.0 mM PMSF, with protease inhibitors (Roche)). Protein concentrations were determined using the BCA assay (Beyotime, China). 2.5. Measurement of intracellular ATP by HPLC The MIN6 cells were treated the same way as the section of insulin secretion. After treatment, the samples were prepared using the procedure described by Giovanni Manfredi et al. [16]. According to the chromatography conditions as described by Fan et al., [17], 20 μL aliquots of the sample were injected into the chromatographical column and were eluted with a buffer containing 100 mM phosphate buffer and 5 mM tetrabutylammonium (pH 6.5) at 1.0 mL/min. ATP Na2 was purchased from SIGMA. Under these conditions, separation of peaks for ATP eluted at 27 min, after injection into the column, the peak heights are linearly proportional to the amount injected between 0 and 2 nM for the adenine nucleotides. ATP content measured in total cell lysates was expressed as nmol/mg cellular proteins. Protein concentration was measured using a BCA protein assay kit as recommended by the manufacturer. 2.6. Western blot The MIN6 cells were treated the same way as the section of insulin secretion. According to protein extracting kit instructions, proteins were extracted with the ice cold RIPA buffer containing 1 mM PMSF, 1 mM DTT and protease inhibitor cocktail. Equal amounts of protein (50 μg/lane) were resolved by 12% SDSpolyacrylamide gel electrophoreses (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, MA). The membrane was further incubated with GLUT2 (1:1000) antibodies (Santa Cruz Biotechnology). The membrane was continuously incubated with appropriate secondary antibodies coupled to horseradish peroxidase and developed in the ECL western detection reagents. The immunoreactive bands were visualized by an enhanced chemiluminescence and then were quantified by a densitometric analysis. 2.7. Statistical analysis The data are presented as mean ± SEM. Statistical significance of differences was analyzed by one-way analysis of variance (ANOVA). All statistics was calculated with software SPSS 13.0 (SPSS Inc., USA). The significance level for all comparisons was P b 0.05. 3. Results 3.1. Biotransformation of Rd to CK Ginsenoside Rd was isolated from ginseng roots by the previously reported method [15]. The snailase was used to convert Rd to CK under optimized conditions. In brief, the snailase was incubated with Rd in a pH 4.5 sodium acetate buffer with agitation at a temperature of 50 °C and enzyme load of 20% for a reaction time of 24 h. The mixture was subsequently placed in a water bath at 90 °C to terminate the enzymatic reaction. The reaction mixtures were individually evaporated, dissolved in methanol, and separated through repeated silica column chromatography to obtain CK. Fig. 1 showed the chromatograms of Rd before and after enzymatic preparation. J. Gu et al. / Fitoterapia 87 (2013) 84–88 85 3.2. CK's effect on MIN6 cell viability To avoid excessive cytotoxicity at high doses, we firstly evaluated the possible cytotoxicity of CK on MIN6 cells at various concentrations (2, 4, 8, 16, and 32 μM) for 24 h. The cell viability, expressed as percentage of the untreated control (100% cell viability), was investigated by MTT assay. As shown in Fig. 2, under the concentration 16 μM, CK had little effect on the viability of MIN6 cells. But when the concentrations reach 32 μM, CK decreased the cell viability to around 83%. Hence we chose the safe concentration of 8 μM for the following experiment. 3.3. CK's effect on insulin secretion In our previous study, we have confirmed that CK treatment increased the plasma insulin levels in the HFD/STZ mouse model [18]. Moreover, others have reported CK's acute insulin secretagogues effect on HIT-T15 cells. Therefore, we further investigate the CK's long term influence on the insulin secretion by glucose-stimulated insulin secretion (GSIS). After a 24-hour incubation with CK, MIN6 cells were exposed to the low (3 mM) and stimulatory (30 mM) glucose concentrations. As shown in Fig. 3, insulin level was higher in stimulatory glucose concentration than in low concentration, CK-8 μM enhanced insulin secretion under stimulatory glucose concentration (P b 0.05). 3.4. CK's effect on the expression of GLUT2 Since glucose metabolism may be involved in the CK's secretagogue effect, we next analyzed GLUT2 protein levels which are a glucose sensor in pancreatic ß-cells. As determined by western blot analysis on MIN6 cells (Fig. 4), the expression of GLUT2 in CK-treated MIN6 cells was significantly upregulated under high glucose (P b 0.05). 3.5. CK's effect on intracellular ATP concentration ATP produced by glucose metabolism in mitochondria is essential for insulin secretion. Therefore, to explore the mechanism by which CK enhances insulin secretion, intracellular ATP concentrations were measured by high-performance liquid chromatography (HPLC). As shown in Fig. 5, CK treatment at low glucose levels did not change the ATP content in MIN6 cells, compared with control. However, similar to the effect on the expression of GLUT2, the ATP content in CK-treated MIN6 cells was significantly higher than that in control at high glucose concentration (P b 0.05). Fig. 1. HPLC analysis of the bioconversion of ginsenoside Rd to compound K. 0 2 4 8 16 32 0 50 100 150 * CK (µM) Cell viability (% of con) Fig. 2. Effect of CK on cell viability of MIN6 cells. The cells were treated with different concentrations of CK (2, 4, 8, 16, 32 μM) for 24 h, and cell viability was determined by MTT. Data was expressed as means ± SEM (n = 3). ⁎P b 0.05 vs. control group. 86 J. Gu et al. / Fitoterapia 87 (2013) 84–88 4. Discussion For years, the ingredients of ginseng were analyzed by researchers with the modern science and technology, and ginsenosides are found to be one of the main active ingredients of ginseng. More than 40 ginsenosides have been isolated from ginseng roots, among which ginsenosides Rb1, Rg1, Rg3, Re, Rd and Rh1 are studied widely [18]. However, CK does not exist in ginseng. It is a final metabolite of protopanaxadiol ginsenosides, also known as ginsenoside M1, or IH901. Here in this study, we used snailase to convert Rd to CK under optimized conditions. Previous investigations have demonstrated the hypoglycemic effect of ginsenosides Rb1 [19,20], Rb2 [21], Rc [22], Rh2 [23] and Rg3 [24]. Also, CK exhibited acute insulin-secretagogue activities in HIT-T15 cells [8] and reduced hyperglycaemia in db/db mice and HFD/STZ mice model [10]. Consistent with the studies, we found long term exposed CK promoted insulin secretion as well. Although the effect of CK is not remarkable under 3 mmol/L glucose concentration, the stimulation of insulin secretion under 30 mmol/L was significantly higher than that of control. The character of MIN6 cell line may account for this. This cell line is an appropriate model for studying glucose-stimulated insulin secretion [25] and its optimum sugar concentration of culture is 25 mmol/L. Therefore, the response to low glucose may be less sensitive than that to high glucose. This effect of CK is favorable for the treatment of type 2 diabetes, since the risk of hypoglycemia is reduced. GLUT2 is necessary in glucose-stimulated insulin secretion in rodent cell lines [26]. Although it is not the rate-limiting step for insulin secretion [27], low expression of GLUT2 induced spontaneously or experimentally impairs glucose-stimulated secretion, supporting that glucose transporter 2 may exert a permissive action [28]. Therefore, we next examined whether it is associated with the CK's effect on MIN6 cells. We found the expression of GLUT2 under stimulation of 30 mmol/L glucose concentration after CK treatment was significantly higher than that of control. The data indicate that GLUT2 may be a target of CK directly or indirectly. Consistent with the above results, the cellular ATP content produced in glucose metabolism was increased under stimulation of 30 mmol/L glucose concentration after CK treatment. However, the molecular regulation of GLUT2 by CK still needs further studies. Indeed, as observed activity of promoting GLUT4 translocation in 3T3-L1 adipocytes [29], CK may be a mediator for the glucose family members. In addition, previous study from our lab indicated that CK could down-regulate gluconenogenesis key enzyme (G6Papse and PEPCK) which might be one of the hypoglycemic mechanisms of CK treatment [10]. 3mM 30mM 0 200 400 600 800 Contol CK 8 µM * # glucose concentration Insulin (ng/mg protein) Fig. 3. Effect of CK on insulin secretion in MIN6 cells. The cells were preincubated with 8 μM CK for 24 h, and then incubated at low glucose (3 mM) and high glucose (30 mM) for 1 h. Insulin secretion was measured by radioimmunoassay. Data was expressed as means ± SEM (n = 3). ⁎P b 0.05 vs. control group at 30 mM glucose, and #P b 0.05 vs. control group at 3 mM glucose. 3mM 30mM 0 100 200 300 400 Contol CK 8 µM glucose concentration * GLUT2 GAPDH # Relative content (% of control) 60 KD 35 KD Fig. 4. Effect of CK on the expression of GLUT2 in MIN6 cells. The cells were preincubated with 8 μM CK for 24 h, and then incubated at 3 mM glucose concentration and 30 mM glucose concentrations for 1 h. The expression of GLUT2 was measured by western blot. The expression level was normalized to that of GAPDH. Data was expressed as means ± SEM (n = 3). ⁎P b 0.05 vs. control group at 30 mM glucose, and #P b 0.05 vs. control group at 3 mM glucose. 3mM 30mM 0 50 100 150 Contol CK 8 µM glucose concentration * # [ATP] nmol/mg cell protein Fig. 5. Effect of CK on the total cellular ATP in MIN6 cells. The cells were preincubated with 8 μM CK for 24 h, and then incubated at low glucose (3 mM) and high glucose (30 mM) for 1 h. Cells were harvested and lysed immediately in perchloric acid. Total cellular ATP content in cell lysates was measured by HPLC and expressed as nmol ATP/mg cellular proteins. Data was expressed as means ± SEM (n = 3). ⁎P b 0.05 vs. control group at 30 mM glucose, and #P b 0.05 vs. control group at 3 mM glucose. J. Gu et al. / Fitoterapia 87 (2013) 84–88 87 In conclusion, this study has shown that CK exerts prominent stimulatory effects on insulin secretion in the MIN6 cells partly via upregulating the expression of GLUT2. In vivo studies also indicate that the CK decreased blood glucose, increased plasma insulin and improved insulin resistance in HFD/STZ induced type 2 diabetic mice [10]. Taken together, CK exerts anti-diabetic actions through enhancing both insulin sensitivity and insulin secretion. Acknowledgments This work was supported by JiLin department of Health (No. 2011Z060), Jilin Science & Technology Development Plan (2009633, 2012747), General Financial Grant from the China Post-Doctorate Science Foundation (No. 2012M520483). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2013.03.020. References [1] Tawab MA, Bahr U, Karas M, et al. Degradation of ginsenosides in humans after oral administration. Drug Metab Dispos 2003;31(8):1065–71. [2] Wang C-Z, Kim KE, Du G-J, et al. Ultra-performance liquid chromatography and time-of-flight mass spectrometry analysis of ginsenoside metabolites in human plasma. Am J Chin Med 2011;39(06):1161–71. [3] Lee IK, Kang KA, Lim CM, et al. Compound K, a metabolite of ginseng saponin, induces mitochondria-dependent and caspase-dependent apoptosis via the generation of reactive oxygen species in human colon cancer cells. Int J Mol Sci 2010;11(12):4916–31. [4] Kim do Y, Park MW, Yuan HD, et al. Compound K induces apoptosis via CAMK-IV/AMPK pathways in HT-29 colon cancer cells. J Agric Food Chem 2009;57(22):10573–8. [5] Joh EH, Lee IA, Jung IH, et al. Ginsenoside Rb1 and its metabolite compound K inhibit IRAK-1 activation—the key step of inflammation. Biochem Pharmacol 2011;82(3):278–86. [6] Lee HU, Bae EA, Han MJ, et al. Hepatoprotective effect of ginsenoside Rb1 and compound K on tert-butyl hydroperoxide-induced liver injury. Liver Int 2005;25(5):1069–73. [7] Li W, Zhang M, Zheng Y-N, et al. Snailase preparation of ginsenoside M1 from protopanaxadiol-type ginsenoside and their protective effects against CCl4-induced chronic hepatotoxicity in mice. Molecules 2011;16(12): 10093–103. [8] Han GC, Ko SK, Sung JH, et al. Compound K enhances insulin secretion with beneficial metabolic effects in db/db mice. J Agric Food Chem 2007;55(26):10641–8. [9] Yoon SH, Han EJ, Sung JH, et al. Anti-diabetic effects of compound K versus metformin versus compound K-metformin combination therapy in diabetic db/db mice. Biol Pharm Bull 2007;30(11):2196–200. [10] Li W, Zhang M, Gu J, et al. Hypoglycemic effect of protopanaxadiol-type ginsenosides and compound K on type 2 diabetes mice induced by high-fat diet combining with streptozotocin via suppression of hepatic gluconeogenesis. Fitoterapia 2012;83(1):192–8. [11] Schuit FC, Huypens P, Heimberg H, et al. Glucose sensing in pancreatic beta-cells: a model for the study of other glucose-regulated cells in gut, pancreas, and hypothalamus. Diabetes 2001;50(1):1–11. [12] Leturque A, Brot-Laroche E Le, Gall M. GLUT2 mutations, translocation, and receptor function in diet sugar managing. Am J Physiol Endocrinol Metab 2009;296(5):E985–92. [13] Matschinsky FM, Glaser B, Magnuson MA. Pancreatic beta-cell glucokinase: closing the gap between theoretical concepts and experimental realities. Diabetes 1998;47(3):307–15. [14] Wang Z, Gleichmann H. GLUT2 in pancreatic islets: crucial target molecule in diabetes induced with multiple low doses of streptozotocin in mice. Diabetes 1998;47(1):50–6. [15] Liu R, Zhang J, Liu W, et al. Anti-obesity effects of protopanaxdiol types of ginsenosides isolated from the leaves of American ginseng (Panax quinquefolius L.) in mice fed with a high-fat diet. Fitoterapia 2010;81(8): 1079–87. [16] Manfredi G, Yang L, Gajewski CD, et al. Measurements of ATP in mammalian cells. Methods 2002;26(4):317–26. [17] Fan SH, Xing LL, Guan YX. Detection of energy metabolism in biological tissues and cells by reversed-phase high performance liquid chromatography. J Clin Rehabil Tissue Eng Res 2008;12(34):6746–8. [18] Lu JM, Yao Q, Chen C. Ginseng compounds: an update on their molecular mechanisms and medical applications. Curr Vasc Pharmacol 2009;7(3): 293–302. [19] Shang W, Yang Y, Zhou L, et al. Ginsenoside Rb1 stimulates glucose uptake through insulin-like signaling pathway in 3T3-L1 adipocytes. J Endocrinol 2008;198(3):561–9. [20] Xiong Y, Shen L, Liu KJ, et al. Antiobesity and antihyperglycemic effects of ginsenoside Rb1 in rats. Diabetes 2010;59(10):2505–12. [21] Yokozawa T, Kobayashi T, Oura H, et al. Studies on the mechanism of the hypoglycemic activity of ginsenoside-Rb2 in streptozotocin-diabetic rats. Chem Pharm Bull (Tokyo) 1985;33(2):869–72. [22] Lee MS, Hwang JT, Kim SH, et al. Ginsenoside Rc, an active component of Panax ginseng, stimulates glucose uptake in C2C12 myotubes through an AMPK-dependent mechanism. J Ethnopharmacol 2010;127(3):771–6. [23] Lee WK, Kao ST, Liu IM, et al. Increase of insulin secretion by ginsenoside Rh2 to lower plasma glucose in Wistar rats. Clin Exp Pharmacol Physiol 2006;33(1–2):27–32. [24] Kim M, Ahn BY, Lee JS, et al. The ginsenoside Rg3 has a stimulatory effect on insulin signaling in L6 myotubes. Biochem Biophys Res Commun 2009;389(1):70–3. [25] Ishihara H, Asano T, Tsukuda K, et al. Pancreatic beta cell line MIN6 exhibits characteristics of glucose metabolism and glucose-stimulated insulin secretion similar to those of normal islets. Diabetologia 1993;36(11):1139–45. [26] Motoyoshi S, Shirotani T, Araki E, et al. Cellular characterization of pituitary adenoma cell line (AtT20 cell) transfected with insulin, glucose transporter type 2 (GLUT2) and glucokinase genes: insulin secretion in response to physiological concentrations of glucose. Diabetologia 1998;41(12):1492–501. [27] Frese T, Bazwinsky I, Muhlbauer E, et al. Circadian and age-dependent expression patterns of GLUT2 and glucokinase in the pancreatic beta-cell of diabetic and nondiabetic rats. Horm Metab Res 2007;39(8):567–74. [28] Cunha AC, Pereira RO, Pereira MJ, et al. Long-term effects of overfeeding during lactation on insulin secretion—the role of GLUT-2. J Nutr Biochem 2009;20(6):435–42. [29] Huang Y-C, Lin C-Y, Huang S-F, et al. Effect and mechanism of ginsenosides CK and Rg1 on stimulation of glucose uptake in 3T3-L1 adipocytes. J Agric Food Chem 2010;58(10):6039–47. 88 J. Gu et al. / Fitoterapia 87 (2013) 84–88

Комментировать