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The insulin-suppressive effect of resveratrol — An in vitro and in vivo phenomenon
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Life Sciences 82 (2008) 430 – 435 www.elsevier.com/locate/lifescie
The insulin-suppressive effect of resveratrol — An in vitro and in vivo phenomenon Tomasz Szkudelski ⁎ Department of Animal Physiology and Biochemistry, August Cieszkowski University of Agriculture, 60-637 Wolynska 35, Poznan, Poland Received 19 July 2007; accepted 1 December 2007
Abstract Resveratrol, a naturally occurring phytoalexin, is known to exert numerous beneficial effects in the organism. Literature data indicate that this compound may, among other effects, play a role in prevention of diabetes and diabetic complications. Resveratrol was recently found to affect insulin secretion in vitro and to change blood insulin concentrations. These effects are, however, not fully elucidated. In the present study, 1, 10 and 100μM resveratrol incubated for 90min with pancreatic islets isolated from normal rats failed to affect basal insulin release, but substantially impaired the secretory response to physiological and maximally effective glucose. In depolarized islets exposed to resveratrol, succinate-induced insulin secretion was also diminished. The blockade of somatostatin receptors substantially enhanced insulin secretion induced by 6.7mM glucose and simultaneously suppressed the inhibitory effect of 1μM resveratrol, but at 10 and 100μM, resveratrol was still able to attenuate hormone secretion. Acetylcholine clearly increased the insulin-secretory response to 6.7mM glucose and canceled the inhibitory effect of 1μM resveratrol. However, resveratrol at concentrations 10 and 100μM strongly decreased insulin secretion. The direct activation of protein kinase C totally suppressed the inhibitory influence of 1 and 10μM resveratrol on hormone secretion. However, activation of this enzyme appeared to be insufficient to cancel the insulin-suppressive effect of 100μM resveratrol. These data indicate that resveratrol-induced inhibition of insulin secretion may be partially mitigated by suppression of somatostatin action, activation of protein kinase C or the presence of acetylcholine. The in vivo experiment revealed that resveratrol, administered to normal rats at the dose 50mg/kg body weight, diminished blood insulin concentrations at 30min, without concomitant changes in glycemia. These observations point to the direct insulin-suppressive action of resveratrol in the rat. © 2007 Elsevier Inc. All rights reserved. Keywords: Resveratrol; Insulin secretion; Pancreatic islets; Insulinemia
Introduction More than 30years ago, Greenwood et al. (1976) hypothesized that disturbed insulin-secretory response in diabetics arises from chronic overstimulation of β-cells. According to this assumption, a temporary β-cell rest should, at least partially, ameliorate their endocrine function in the future. Results of many studies have confirmed that this is indeed the case (reviewed by Hansen et al., 2004). Beneficial effects of β-cell rest were found in patients with type 1 diabetes (Björk et al., 1996), in rats with type 1 diabetes model (Matsuda et al., 2002; Skak et al., 2004) and in pancreatectomized animals (Leahy et al., 1994). Moreover, in humans with type 2 diabetes, prevention of ⁎ Tel.: +48 61 8466083; fax: +48 61 8487197. E-mail address: [email protected]. 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.12.008
β-cell exhaustion resulted afterwards in ameliorated secretion of the pancreatic hormone (Laedtke et al., 2000, Guldstrand et al., 2002). The progress of type 2 diabetes was found to be delayed in rodents treated with inhibitors of insulin secretion (Leahy et al., 1994; Surwit et al., 2000; Alemzadeh et al., 2004). Beneficial effects of insulin-suppressive agents were also demonstrated in obese Zucker rats (Alemzadeh et al., 2004). Furthermore, some problems resulting from exaggerated secretion of insulin appearing in patients with polycystic ovary syndrome (Welt et al., 2002), insulinoma (von Eyben et al., 1994; Tanaka et al., 2000) or persistent hyperinsulinemic hypoglycemia of infancy (nesidioblastosis) (Kane et al., 1997; Meissner et al., 1997) are partially resolved by insulin-suppressive drugs. In clinical practice, only a few inhibitors of insulin secretion are used: two antagonists of K+-ATP channels (diazoxide and NN414) and one somatostatin analogue (octreotide). However,
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chronic administration of these agents causes some side effects (Björk et al., 1996; Cosgrove et al., 2002). An insulin-suppressive drug that can be administered for a long time without any unfavorable effects is still lacking. Resveratrol (3, 5, 4′-trihydroxystilbene; Fig. 1) is a naturally occurring phytoalexin exerting numerous beneficial effects in the organism. The most recent studies revealed that resveratrol is able to reduce many consequences of a high-calorie diet in mice and to increase their survival (Baur et al., 2006). Moreover, mice on a high-fat diet consuming resveratrol remained lean and had improved muscle parameters (Lagouge et al., 2006). Interestingly, in both cases resveratrol protected animals against insulin resistance (Baur et al., 2006; Lagouge et al., 2006). An insulin-like effect of resveratrol was also noticed in diabetic rats (Su et al., 2006; Chi et al., 2007). On the other hand, it was recently found that resveratrol decreased insulin secretion from pancreatic islets of normal rats (Szkudelski, 2006, 2007) and was able to affect blood insulin concentrations (Baur et al., 2006; Su et al., 2006; Chen et al., 2007; Chi et al., 2007). These effects are, however, not fully elucidated and some results are contradictory. It is worth noting that considerable amounts of resveratrol are present, for example, in red wine (Siemann and Creasy, 1992), and the pure compound is available in tablets and is recommended as a dietary supplement. Therefore, it seems that its influence on various processes in the organism should be precisely defined. The purpose of the present study was to determine of some aspects of resveratrol's influence on insulin secretion from rat pancreatic islets. Moreover, the short-term effects of resveratrol on blood insulin and glucose concentrations in normal rats were ascertained. Materials and methods Animals In all experiments male Wistar rats weighing 200–260g (insulin secretion) or 140–160g (the in vivo experiment) obtained from Brwinow (Poland) were used. Animals were kept in cages in an air-conditioned animal room in standard conditions, at constant temperature (21 ± 1 °C) with a 12:12-h dark-light cycle and were fed ad libitum a complete laboratory diet Labofeed B (Kcynia, Poland). The protocols of all experiments were accepted by the Local Ethical Commission for Investigation on Animals. In vitro experiments Islet isolation In each experiment, islets were isolated from three rats by a collagenase digestion technique (Lacy and Kostianovsky, 1967).
Fig. 1. The chemical structure of trans-resveratrol.
Fig. 2. Effects of resveratrol on insulin secretion from rat pancreatic islets exposed to 2.8 (upper plot), 6.7 (middle plot) or 30 (lower plot) mM glucose. Islets were incubated for 90 min in Krebs–Ringer buffer at different concentrations of resveratrol. Values represent means ± SEM of 12 determinations from three separate experiments. Means marked by different letters differ statistically at P b 0.05.
Hanks solution saturated with a mixture of O2/CO2 (95%/5%) was used during isolation. The isolated glands were pulled, cut with scissors, and incubated with collagenase. Then, the tissue was washed several times with Hanks' solution without collagenase. Islets were separated from the remaining exocrine tissue by hand picking under a stereomicroscope. Insulin secretion from isolated islets Groups of 5 islets were incubated in 1ml of Krebs–Ringer buffer (pH = 7.4) for 90min at 37°C in an atmosphere of O2/CO2 (95%/5%) with a gentle shaking. In each experiment, the concentration of resveratrol in the buffer was 1, 10 and 100μM. Resveratrol at these concentrations was previously found to effectively reduce insulin secretion (Szkudelski, 2006, 2007). Simultaneously, control incubations without the tested compound were performed. It was previously demonstrated that under these conditions resveratrol did not induce permanent, harmful effects in pancreatic islets (Szkudelski, 2006, 2007).
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In vivo experiment Animals were divided into three groups consisting of 16 rats each. Rats in the first (control) group received vehicle i.e. dimethyl sulfoxide: water mixture (4: 6v/v; 5ml/kg body weight) whereas rats in the second and third group received vehicle with 10 and 50mg resveratrol/kg body weight, respectively. The vehicle and resveratrol solutions were administered intragastrically and 30min later animals were killed by decapitation, their blood serum was separated and stored (− 80 °C) until analysis. The time 30min was chosen according to results of Juan et al. (2002) demonstrating that in rats resveratrol is quickly absorbed and reaches a maximum in the blood 10min after oral administration. The experiment was preceded by a five-day intragastrical water administration to minimize the stress caused by animal treatment. Blood glucose and insulin determinations
Fig. 3. Effects of resveratrol on succinate-induced insulin secretion from depolarized islets (upper plot) and on glucose-induced insulin secretion in the presence of cyclosomatostatin (lower plot). Islets were incubated for 90 min in Krebs–Ringer buffer containing 250 μM diazoxide and 30 mM K+ without succinate (upper plot, gray bar) or with 10 mM succinate at different concentrations of resveratrol (upper plot, black bars). To determine the effect of resveratrol on insulin secretion in the presence of cyclosomatostatin, islets were incubated for 90 min in Krebs–Ringer buffer containing 6.7 mM glucose (lower plot, grey bar) or 6.7 mM glucose and 10 μM cyclosomatostatin at different concentrations of resveratrol (lower plot, black bars). Values represent means ± SEM of 12 determinations from three separate experiments. Means marked by different letters differ statistically at P b 0.05.
To determine the effects of resveratrol on basal, physiological and maximally effective insulin secretion, pancreatic islets were incubated with or without resveratrol in the presence of 2.8, 6.7 and 30mM glucose, respectively. In further investigations, the effects of resveratrol on insulin secretion induced by 10mM succinate in depolarized islets were tested. To depolarize plasma membrane, islets were exposed to 30mM K+ and 0.25mM diazoxide, whereas the concentration of NaCl in Krebs–Ringer buffer was diminished to maintain the osmolarity. In order to ascertain whether resveratrol affects glucoseinduced insulin secretion under conditions of activated protein kinase C (PKC), pancreatic islets were stimulated with 6.7mM glucose in the presence of 500nM phorbol 12-myristate 13acetate (PMA), an activator of the enzyme. Moreover, the effects of resveratrol on glucose-induced (6.7mM) insulin secretion in the presence of 10μM acetylcholine were studied. The effects of resveratrol on the insulin-secretory response to 6.7mM glucose were also tested when islet somatostatin receptors were blocked by 10μM cyclosomatostatin. At the end of each experiment, the incubation medium was immediately sampled and stored (− 80 °C) until insulin determination.
The blood serum glucose was determined by the glucose oxidase method (Bergmeyer and Bernt, 1974). Insulin in blood serum and insulin released from isolated islets was measured by radioimmunoassay using kits specific for rat hormone (Linco Research, Inc., USA).
Fig. 4. Effects of resveratrol on glucose-induced insulin secretion from rat pancreatic islets in the presence of acetylcholine (upper plot) or PMA (lower plot). Islets were incubated for 90 min in Krebs–Ringer buffer containing 6.7 mM glucose alone (gray bars), glucose with 10 μM acetylcholine (upper plot, black bars) or glucose with 500 nM PMA (lower plot, black bars) at different concentrations of resveratrol. Values represent means ± SEM of 12 determinations from three separate experiments. Means marked by different letters differ statistically at P b 0.05.
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Reagents Trans-Resveratrol (obtained via extraction from plant Bushy Knotweed), D-glucose, phorbol 12-myristate 13-acetate (PMA), mono-methyl succinate, acetylcholine chloride, cyclosomatostatin, bovine serum albumin (fatty acid free) and all reagents used to prepare Hanks' solution and Krebs–Ringer buffer and to determine blood glucose were obtained from Sigma. Dimethyl sulfoxide was from ICN Biomedicals Inc., whereas collagenase P (from Clostridium histolyticum) was purchased from Roche Diagnostics. The composition of Hanks' solution used for islet isolation was the following (in mM): NaCl 137, KCl 5.36, MgSO4 0.81, Na2HPO4 0.34, KH2PO4 0.44, CaCl2 1.26, NaHCO3 4.17. Krebs–Ringer buffer applied during incubations consisted of (in mM): NaCl 115, NaHCO3 24, KCl 5, MgCl2 1, CaCl2 1 and 0.5% (w/v) bovine serum albumin. Stock solutions of resveratrol, and PMA were prepared in dimethyl sulfoxide. The concentration of the solvent in the buffer with islets was less that 0.1%.
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Table 1 The effect of resveratrol on blood glucose, insulin and glucose/insulin ratio in normal rats Parameter
Control
Resveratrol 10 mg/kg body weight
Resveratrol 50 mg/kg body weight
Glucose (mM) Insulin (ng/ml) Glucose/insulin ratio (mM/ng/ml)
5.650 ± 0.262 1.345 ± 0.117 4.200 ± 0.299
6.067 ± 0.452 1.304 ± 0.132 4.652 ± 0.301
6.248 ± 0.320 1.027 ± 0.130⁎ 6.083 ± 0.351⁎
Resveratrol was dissolved in dimethyl sulfoxide:water mixture (4:6 v/v; 0.5 ml/ 100 g body weight) and was given intragastrically as a single dose 30 min before decapitation. Values represents the mean (n = 16) ± SEM. ⁎P b 0.05 vs. control and 10 mg resveratrol.
Means obtained in each group of rats (in vivo experiment) and from three separate experiments (in quadruplicates) in the case of insulin secretion studies were evaluated statistically using the analysis of variance and Duncan's multiple range test. Differences were considered significant at P b 0.05.
glucose. Acetylcholine increased hormone secretion by 130%. Under these stimulatory conditions, the influence of 1μM resveratrol on insulin secretion was negligible. However, despite the presence of acetylcholine, 10 and 100μM resveratrol still diminished secretion of insulin by 37% and 50%, respectively (Fig. 4). The direct activation of PKC by 500nM PMA in islets incubated with 6.7mM glucose resulted in a substantial increase (by 250%) in insulin secretion compared with islets exposed to glucose alone. The presence of 500nM PMA completely canceled the inhibitory effect of 1 and 10μM resveratrol, but resveratrol at the highest concentration (100μM) was still able to reduce (by 25%) the secretory response of β-cells (Fig. 4).
Results
In vivo experiment
In vitro experiments
Resveratrol administered to rats at the dose 10 or 50mg/kg body weight did not significantly affect blood glucose. The influence of resveratrol administered at low dose on blood insulin concentrations was also negligible. However, in rats receiving 50mg resveratrol/kg body weight a 24% decrease in insulinemia was found. Glucose/insulin ratio increased significantly (by 45%) only in animals treated with resveratrol at the dose 50mg/kg (Table 1).
Statistical analysis
Experiments with isolated pancreatic islets incubated for 90min with resveratrol revealed that basal release of insulin studied in the presence of 2.8mM glucose was unchanged by 1, 10 and 100μM resveratrol. However, insulin secretion induced by 6.7mM glucose was substantially reduced in islets exposed to resveratrol (1, 10 and 100μM). Similarly, maximal secretory response to 30mM glucose was clearly affected by the tested compound. In the presence of 1μM resveratrol insulin secretion tended to be reduced, but the effect was not statistically significant. Islets stimulated with 30mM glucose and exposed to 10 and 100μM resveratrol secreted significantly less insulin, by 20% and 30%, respectively, compared with untreated islets (Fig. 2). In depolarized islets, 10mM succinate enhanced insulin secretion by 380% compared with basal release. The insulinotropic action of this secretagogue was substantially blunted in the presence of 1, 10 and 100μM resveratrol by 24%, 30% and 25%, respectively (Fig. 3). The blockade of somatostatin receptors in islets stimulated with 6.7mM glucose resulted in increased secretion of insulin by 250% compared with glucose alone. Under these conditions, 1μM resveratrol failed to alter hormone secretion. However, islets exposed to 10 and 100μM resveratrol released less insulin by 18% and 40% than islets incubated without the tested compound (Fig. 3). Further studies demonstrated that in pancreatic islets 10μM acetylcholine potentiated the insulin-secretory response to 6.7mM
Discussion Results obtained in this study and in experiments performed previously (Szkudelski, 2006) clearly demonstrated that resveratrol incubated with rat pancreatic islets attenuated insulin secretion induced by glucose at physiological, supraphysiological and maximally effective concentrations, whereas basal hormone release remained unchanged. This indicates that the tested compound evinces its insulin-suppressive activity at a broad spectrum of stimulatory glucose concentrations ranging from 6.7 to 30 mM, but is completely ineffective at the non-stimulatory one. The lack of effects at the non-stimulatory concentration of glucose and a clearcut inhibition of secretion stimulated by succinate, a secretagogue metabolized exclusively in the mitochondria (Fahien and MacDonald, 2002), are in agreement with previous findings and provide further evidence confirming that the attenuation of insulin secretion caused by resveratrol is due to metabolic disturbances in β-cells. These disturbances involved diminished glucose oxidation, enhanced lactate production and reduced ATP levels. Moreover,
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glucose-induced hyperpolarization of mitochondrial membrane was attenuated in the presence of resveratrol (Szkudelski, 2007). The direct inhibitory effect of resveratrol on ATP synthase in the mitochondria isolated from rat brain and liver was also demonstrated (Zini et al., 1999; Zheng and Ramirez, 2000). On the other hand, Chen et al. (2007) recently showed that resveratrol increased insulin secretion from different β-cell insulinoma lines (MIN6, Hit-T15 and RIN-m5F). Such a distinction between whole islets and isolated β-cells could partially result from paracrine interactions within islets involving augmented secretion of somatostatin (Schatz and Kullek, 1980; Samols et al., 1986). The suppression of somatostatin action in islets canceled the inhibitory influence of 1 µM resveratrol. However, despite the blockade of somatostatin receptors, the insulin-suppressive effect of 10 and 100 µM resveratrol was still present. Since these observations were made in islets stimulated with 6.7 mM glucose, one can suppose that reduced secretion of insulin resulted from resveratrol-induced disturbances in metabolism of glucose. The most recent data provide evidence that resveratrol binds to SUR1, effectively displaces glibenclamide and blocks K+-ATP channels in mouse β-cells (Hambrock et al., 2007). The inhibitory influence of the tested compound on K+ current via these channels with a concomitant depolarization of plasma membrane was also shown in MIN6 cells (Chen et al., 2007). These effects should contribute to increased secretion of insulin. However, in experiments with freshly isolated rat pancreatic islets the opposite effects were found. Resveratrol reduced hormone secretion not only upon stimulation with glucose alone, but also in the presence of glibenclamide (Szkudelski, 2006) and under conditions when K+-ATP channels were bypassed (Szkudelski, 2007). Moreover, inhibition of succinate-induced insulin release was noted in islets exposed to diazoxide and high potassium to depolarize the plasma membrane. These secretory data allow the conclusion that interactions of resveratrol with K+-ATP channels are not involved in the attenuation of the endocrine function of rat β-cells, at least under experimental conditions applied in the present study and in the previous investigations (Szkudelski, 2006, 2007). Literature data indicate that resveratrol is able to inhibit PKC in different kinds of cells (Atten et al., 2001; Leiro et al., 2002; Woo et al., 2004). This effect is thought to arise from resveratrol's interaction with C1 domains of the enzyme (Slater et al., 2003). Activation of PKC in β-cells is involved in the mechanism of insulin secretion, especially at higher glucose concentrations (Sahai et al., 1992) and upon cholinergic stimulation (Persaud et al., 1989). In pancreatic islets incubated with 6.7 mM glucose and acetylcholine, 1 μM resveratrol failed to affect insulin secretion, but at concentrations 10 and 100 μM substantially aggravated this process. Furthermore, a direct activation of PKC by PMA completely suppressed the inhibitory action of 1 and 10 μM resveratrol on insulin secretion. PMA is usually present at concentration 100 nM to effectively activate PKC in β-cells. In the present study, the concentration of PMA was augmented to 500 nM since the interaction of resveratrol with PKC is competitive with respect to phorbol esters (Slater et al., 2003). However, despite high concentration of PMA, the inhibitory effect of resveratrol on insulin secretion was only partially suppressed. A similar effect was found when islets were exposed to 100 nM
PMA (Szkudelski, 2006). These results demonstrate that the inhibitory action of resveratrol on insulin secretion may be partially mitigated as a result of cholinergic stimulation. One can also suppose that inhibition of PKC activity is involved in the insulin-suppressive action of resveratrol in β-cells. In the light of previous data (Szkudelski, 2006, 2007) and the results of the in vitro experiments obtained in this study, it seems to be important to determine whether resveratrol may affect blood insulin levels. Literature data concerning resveratrol's effects on blood insulin are scanty. Su et al. (2006) revealed that in diabetic rats (a streptozotocin-nicotinamide model) resveratrol administered orally for a few days at a dose as low as 0.5 mg/kg body weight reduced blood insulin. Similarly, in mice with increased blood insulin as a result of a high-calorie diet, resveratrol (0.04% in the diet) also decreased insulinemia (Baur et al., 2006). In the present study, resveratrol administered perorally to normal rats as a single dose of 50 mg/kg body weight (but not at one fifth of that dose) diminished blood insulin. Simultaneously, glucose/insulin ratio increased. Since blood glucose was not significantly affected, one can suppose that the lowered insulinemia was due to the direct inhibitory effect of resveratrol on hormone secretion, as evidenced in vitro. In contrast to these outcomes, it was found that resveratrol administered orally (3 and 10 mg/kg body weight) (Chi et al., 2007) or intraperitoneally (3 mg/kg) (Chen et al., 2007) to normal rats increased blood insulin 20–90 min after administration. A similar effect was noted in diabetic rats with preserved ability to secrete insulin (Chi et al., 2007), but not in rats with type 1 diabetes (Chi et al., 2007; Chen et al., 2007). Data from the literature and the results of the present study indicate that doses of resveratrol necessary to induce changes in blood insulin are varied. However, these amounts are considerably below the toxic ones shown by Hebbar et al. (2005). In that study, rats were given 300, 1000 or 3000 mg resveratrol per kg body weight per day for four weeks; some harmful effects (nephrotoxicity, mild liver toxicity, moderate changes in enzyme activities and gene expression) were seen in rats receiving 1000 and 3000 mg resveratrol, but not in rats receiving 300 mg. In conclusion, results obtained in these studies demonstrated that resveratrol incubated with pancreatic islets isolated from normal rats did not affect basal insulin release, but clearly restricted physiological and maximal insulin-secretory response to glucose. The lack of influence on non-stimulated hormone release can be considered as a positive finding. It was also revealed that the inhibition of insulin secretion in islets exposed to resveratrol may be partially mitigated by acetylcholine, direct activation of PKC or blocking of somatostatin receptors. However, under these conditions resveratrol was still able to reduce insulin secretion, probably due to the inhibition of glucose metabolism. This is in accord to observations demonstrating that resveratrol does not disturb hormone secretion when this process is induced without metabolic events (Szkudelski, 2007). The obtained results strongly support the previous data that resveratrol exerts a clearcut insulin-suppressive action in rat pancreatic islets. Moreover, the experiment in vivo demonstrated that resveratrol is able to diminish blood insulin in normal rats provided that an appropriately large amount of this compound is administered. However, further studies are required to clarify resveratrol's
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Lagouge, M., Argmann, C., Gerhart-Hines, Z., Meziane, H., Lerin, C., Daussin, F., Messadeq, N., Milne, J., Lambert, P., Elliott, P., Geny, B., Laakso, M., Puigserver, P., Auwerx, J., 2006. Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Cell 127 (6), 1109–1122. Leahy, J.L., Bumbalo, L.M., Chen, C., 1994. Diazoxide causes recovery of betacell glucose responsiveness in 90% pancreatectomized diabetic rats. Diabetes 43 (2), 173–179. Leiro, J., Alvarez, E., Garcia, D., Orallo, F., 2002. Resveratrol modulates rat macrophage functions. International Immunopharmacology 2 (6), 767–774. Matsuda, M., Kawasaki, F., Mikami, Y., Takeuchi, Y., Saito, M., Eto, M., Kaku, K., 2002. Rescue of beta-cell exhaustion by diazoxide after the development of diabetes mellitus in rats with streptozotocin-induced diabetes. European Journal of Pharmacology 453 (1), 141–148. Meissner, T., Brune, W., Mayatepek, E., 1997. Persistent hyperinsulinaemic hypoglycaemia of infancy: therapy, clinical outcome and mutational analysis. European Journal of Pediatrics 156 (10), 754–757. Persaud, S.J., Jones, P.M., Sugden, D., Howell, S.L., 1989. The role of protein kinase C in cholinergic stimulation of insulin secretion from rat islets of Langerhans. Biochemical Journal 264 (3), 753–758. Sahai, A., Fadda, G.Z., Massry, S.G., 1992. Parathyroid hormone activates protein kinase C of pancreatic islets. Endocrinology 131 (4), 1889–1894. Samols, E., Bonner-Weir, S., Weir, G.C., 1986. Intra-islet insulin-glucagonsomatostatin relationships. Clinical Endocrinology and Metabolism 15 (1), 33–58. Schatz, H., Kullek, U., 1980. Studies on the local (paracrine) actions of glucagon, somatostatin and insulin in isolated islets of rat pancreas. FEBS Letters 122 (2), 207–210. Siemann, E.H., Creasy, L.L., 1992. Concentration of the phytoalexin resveratrol in wine. American Journal of Enology and Viticulture 43, 49–52. Skak, K., Gotfredsen, C.F., Lundsgaard, D., Hansen, J.B., Sturis, J., Markholst, H., 2004. Improved beta-cell survival and reduced insulitis in a type 1 diabetic rat model after treatment with a beta-cell-selective K(ATP) channel opener. Diabetes 53 (4), 1089–1095. Slater, S.J., Seiz, J.L., Cook, A.C., Stagliano, B.A., Buzas, C.J., 2003. Inhibition of protein kinase C by resveratrol. Biochimica et Biophysica Acta 1637 (1), 59–69. Su, H.C., Hung, L.M., Chen, J.K., 2006. Resveratrol, a red wine antioxidant, possesses an insulin-like effect in streptozotocin-induced diabetic rats. American Journal of Physiology: Endocrinology and Metabolism 290 (6), 1339–1346. Surwit, R.S., Dixon, T.M., Petro, A.E., Daniel, K.W., Collins, S., 2000. Diazoxide restores beta3-adrenergic receptor function in diet-induced obesity and diabetes. Endocrinology 141 (10), 3630–3637. Szkudelski, T., 2006. Resveratrol inhibits insulin secretion from rat pancreatic islets. European Journal of Pharmacology 552 (1–3), 176–181. Szkudelski, T., 2007. Resveratrol-induced inhibition of insulin secretion from rat pancreatic islets: evidence for pivotal role of metabolic disturbances. American Journal of Physiology: Endocrinology and Metabolism 293 (4), 901–907. Tanaka, Y., Funahashi, H., Imai, T., Naruse, T., Suzumura, K., Oda, Y., 2000. The effectiveness of administering a minimal dose of octreotide long-term prior to surgery for insulinoma: report of a case. Surgery Today 30 (6), 541–543. von Eyben, F.E., Grodum, E., Gjessing, H.J., Hagen, C., Nielsen, H., 1994. Metabolic remission with octreotide in patients with insulinoma. Journal of Internal Medicine 235 (3), 245–248. Welt, C.K., Taylor, A.E., Martin, K.A., Hall, J.E., 2002. Serum inhibin B in polycystic ovary syndrome: regulation by insulin and luteinizing hormone. Journal of Clinical Endocrinology and Metabolism 87 (12), 5559–5565. Woo, J.H., Lim, J.H., Kim, Y.H., Suh, S.I., Min do, S., Chang, J.S., Lee, Y.H., Park, J.W., Kwon, T.K., 2004. Resveratrol inhibits phorbol myristate acetateinduced matrix metalloproteinase-9 expression by inhibiting JNK and PKC delta signal transduction. Oncogene 23 (10), 1845–1853. Zheng, J., Ramirez, V.D., 2000. Inhibition of mitochondrial proton F0F1ATPase/ATP synthase by polyphenolic phytochemicals. British Journal of Pharmacology 130 (5), 1115–1123. Zini, R., Morin, C., Bertelli, A., Bertelli, A.A., Tillement, J.P., 1999. Effects of resveratrol on the rat brain respiratory chain. Drugs Under Experimental and Clinical Research 25 (2–3), 87–97.
Life Sciences 82 (2008) 430 – 435 www.elsevier.com/locate/lifescie
The insulin-suppressive effect of resveratrol — An in vitro and in vivo phenomenon Tomasz Szkudelski ⁎ Department of Animal Physiology and Biochemistry, August Cieszkowski University of Agriculture, 60-637 Wolynska 35, Poznan, Poland Received 19 July 2007; accepted 1 December 2007
Abstract Resveratrol, a naturally occurring phytoalexin, is known to exert numerous beneficial effects in the organism. Literature data indicate that this compound may, among other effects, play a role in prevention of diabetes and diabetic complications. Resveratrol was recently found to affect insulin secretion in vitro and to change blood insulin concentrations. These effects are, however, not fully elucidated. In the present study, 1, 10 and 100μM resveratrol incubated for 90min with pancreatic islets isolated from normal rats failed to affect basal insulin release, but substantially impaired the secretory response to physiological and maximally effective glucose. In depolarized islets exposed to resveratrol, succinate-induced insulin secretion was also diminished. The blockade of somatostatin receptors substantially enhanced insulin secretion induced by 6.7mM glucose and simultaneously suppressed the inhibitory effect of 1μM resveratrol, but at 10 and 100μM, resveratrol was still able to attenuate hormone secretion. Acetylcholine clearly increased the insulin-secretory response to 6.7mM glucose and canceled the inhibitory effect of 1μM resveratrol. However, resveratrol at concentrations 10 and 100μM strongly decreased insulin secretion. The direct activation of protein kinase C totally suppressed the inhibitory influence of 1 and 10μM resveratrol on hormone secretion. However, activation of this enzyme appeared to be insufficient to cancel the insulin-suppressive effect of 100μM resveratrol. These data indicate that resveratrol-induced inhibition of insulin secretion may be partially mitigated by suppression of somatostatin action, activation of protein kinase C or the presence of acetylcholine. The in vivo experiment revealed that resveratrol, administered to normal rats at the dose 50mg/kg body weight, diminished blood insulin concentrations at 30min, without concomitant changes in glycemia. These observations point to the direct insulin-suppressive action of resveratrol in the rat. © 2007 Elsevier Inc. All rights reserved. Keywords: Resveratrol; Insulin secretion; Pancreatic islets; Insulinemia
Introduction More than 30years ago, Greenwood et al. (1976) hypothesized that disturbed insulin-secretory response in diabetics arises from chronic overstimulation of β-cells. According to this assumption, a temporary β-cell rest should, at least partially, ameliorate their endocrine function in the future. Results of many studies have confirmed that this is indeed the case (reviewed by Hansen et al., 2004). Beneficial effects of β-cell rest were found in patients with type 1 diabetes (Björk et al., 1996), in rats with type 1 diabetes model (Matsuda et al., 2002; Skak et al., 2004) and in pancreatectomized animals (Leahy et al., 1994). Moreover, in humans with type 2 diabetes, prevention of ⁎ Tel.: +48 61 8466083; fax: +48 61 8487197. E-mail address: [email protected]. 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.12.008
β-cell exhaustion resulted afterwards in ameliorated secretion of the pancreatic hormone (Laedtke et al., 2000, Guldstrand et al., 2002). The progress of type 2 diabetes was found to be delayed in rodents treated with inhibitors of insulin secretion (Leahy et al., 1994; Surwit et al., 2000; Alemzadeh et al., 2004). Beneficial effects of insulin-suppressive agents were also demonstrated in obese Zucker rats (Alemzadeh et al., 2004). Furthermore, some problems resulting from exaggerated secretion of insulin appearing in patients with polycystic ovary syndrome (Welt et al., 2002), insulinoma (von Eyben et al., 1994; Tanaka et al., 2000) or persistent hyperinsulinemic hypoglycemia of infancy (nesidioblastosis) (Kane et al., 1997; Meissner et al., 1997) are partially resolved by insulin-suppressive drugs. In clinical practice, only a few inhibitors of insulin secretion are used: two antagonists of K+-ATP channels (diazoxide and NN414) and one somatostatin analogue (octreotide). However,
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chronic administration of these agents causes some side effects (Björk et al., 1996; Cosgrove et al., 2002). An insulin-suppressive drug that can be administered for a long time without any unfavorable effects is still lacking. Resveratrol (3, 5, 4′-trihydroxystilbene; Fig. 1) is a naturally occurring phytoalexin exerting numerous beneficial effects in the organism. The most recent studies revealed that resveratrol is able to reduce many consequences of a high-calorie diet in mice and to increase their survival (Baur et al., 2006). Moreover, mice on a high-fat diet consuming resveratrol remained lean and had improved muscle parameters (Lagouge et al., 2006). Interestingly, in both cases resveratrol protected animals against insulin resistance (Baur et al., 2006; Lagouge et al., 2006). An insulin-like effect of resveratrol was also noticed in diabetic rats (Su et al., 2006; Chi et al., 2007). On the other hand, it was recently found that resveratrol decreased insulin secretion from pancreatic islets of normal rats (Szkudelski, 2006, 2007) and was able to affect blood insulin concentrations (Baur et al., 2006; Su et al., 2006; Chen et al., 2007; Chi et al., 2007). These effects are, however, not fully elucidated and some results are contradictory. It is worth noting that considerable amounts of resveratrol are present, for example, in red wine (Siemann and Creasy, 1992), and the pure compound is available in tablets and is recommended as a dietary supplement. Therefore, it seems that its influence on various processes in the organism should be precisely defined. The purpose of the present study was to determine of some aspects of resveratrol's influence on insulin secretion from rat pancreatic islets. Moreover, the short-term effects of resveratrol on blood insulin and glucose concentrations in normal rats were ascertained. Materials and methods Animals In all experiments male Wistar rats weighing 200–260g (insulin secretion) or 140–160g (the in vivo experiment) obtained from Brwinow (Poland) were used. Animals were kept in cages in an air-conditioned animal room in standard conditions, at constant temperature (21 ± 1 °C) with a 12:12-h dark-light cycle and were fed ad libitum a complete laboratory diet Labofeed B (Kcynia, Poland). The protocols of all experiments were accepted by the Local Ethical Commission for Investigation on Animals. In vitro experiments Islet isolation In each experiment, islets were isolated from three rats by a collagenase digestion technique (Lacy and Kostianovsky, 1967).
Fig. 1. The chemical structure of trans-resveratrol.
Fig. 2. Effects of resveratrol on insulin secretion from rat pancreatic islets exposed to 2.8 (upper plot), 6.7 (middle plot) or 30 (lower plot) mM glucose. Islets were incubated for 90 min in Krebs–Ringer buffer at different concentrations of resveratrol. Values represent means ± SEM of 12 determinations from three separate experiments. Means marked by different letters differ statistically at P b 0.05.
Hanks solution saturated with a mixture of O2/CO2 (95%/5%) was used during isolation. The isolated glands were pulled, cut with scissors, and incubated with collagenase. Then, the tissue was washed several times with Hanks' solution without collagenase. Islets were separated from the remaining exocrine tissue by hand picking under a stereomicroscope. Insulin secretion from isolated islets Groups of 5 islets were incubated in 1ml of Krebs–Ringer buffer (pH = 7.4) for 90min at 37°C in an atmosphere of O2/CO2 (95%/5%) with a gentle shaking. In each experiment, the concentration of resveratrol in the buffer was 1, 10 and 100μM. Resveratrol at these concentrations was previously found to effectively reduce insulin secretion (Szkudelski, 2006, 2007). Simultaneously, control incubations without the tested compound were performed. It was previously demonstrated that under these conditions resveratrol did not induce permanent, harmful effects in pancreatic islets (Szkudelski, 2006, 2007).
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In vivo experiment Animals were divided into three groups consisting of 16 rats each. Rats in the first (control) group received vehicle i.e. dimethyl sulfoxide: water mixture (4: 6v/v; 5ml/kg body weight) whereas rats in the second and third group received vehicle with 10 and 50mg resveratrol/kg body weight, respectively. The vehicle and resveratrol solutions were administered intragastrically and 30min later animals were killed by decapitation, their blood serum was separated and stored (− 80 °C) until analysis. The time 30min was chosen according to results of Juan et al. (2002) demonstrating that in rats resveratrol is quickly absorbed and reaches a maximum in the blood 10min after oral administration. The experiment was preceded by a five-day intragastrical water administration to minimize the stress caused by animal treatment. Blood glucose and insulin determinations
Fig. 3. Effects of resveratrol on succinate-induced insulin secretion from depolarized islets (upper plot) and on glucose-induced insulin secretion in the presence of cyclosomatostatin (lower plot). Islets were incubated for 90 min in Krebs–Ringer buffer containing 250 μM diazoxide and 30 mM K+ without succinate (upper plot, gray bar) or with 10 mM succinate at different concentrations of resveratrol (upper plot, black bars). To determine the effect of resveratrol on insulin secretion in the presence of cyclosomatostatin, islets were incubated for 90 min in Krebs–Ringer buffer containing 6.7 mM glucose (lower plot, grey bar) or 6.7 mM glucose and 10 μM cyclosomatostatin at different concentrations of resveratrol (lower plot, black bars). Values represent means ± SEM of 12 determinations from three separate experiments. Means marked by different letters differ statistically at P b 0.05.
To determine the effects of resveratrol on basal, physiological and maximally effective insulin secretion, pancreatic islets were incubated with or without resveratrol in the presence of 2.8, 6.7 and 30mM glucose, respectively. In further investigations, the effects of resveratrol on insulin secretion induced by 10mM succinate in depolarized islets were tested. To depolarize plasma membrane, islets were exposed to 30mM K+ and 0.25mM diazoxide, whereas the concentration of NaCl in Krebs–Ringer buffer was diminished to maintain the osmolarity. In order to ascertain whether resveratrol affects glucoseinduced insulin secretion under conditions of activated protein kinase C (PKC), pancreatic islets were stimulated with 6.7mM glucose in the presence of 500nM phorbol 12-myristate 13acetate (PMA), an activator of the enzyme. Moreover, the effects of resveratrol on glucose-induced (6.7mM) insulin secretion in the presence of 10μM acetylcholine were studied. The effects of resveratrol on the insulin-secretory response to 6.7mM glucose were also tested when islet somatostatin receptors were blocked by 10μM cyclosomatostatin. At the end of each experiment, the incubation medium was immediately sampled and stored (− 80 °C) until insulin determination.
The blood serum glucose was determined by the glucose oxidase method (Bergmeyer and Bernt, 1974). Insulin in blood serum and insulin released from isolated islets was measured by radioimmunoassay using kits specific for rat hormone (Linco Research, Inc., USA).
Fig. 4. Effects of resveratrol on glucose-induced insulin secretion from rat pancreatic islets in the presence of acetylcholine (upper plot) or PMA (lower plot). Islets were incubated for 90 min in Krebs–Ringer buffer containing 6.7 mM glucose alone (gray bars), glucose with 10 μM acetylcholine (upper plot, black bars) or glucose with 500 nM PMA (lower plot, black bars) at different concentrations of resveratrol. Values represent means ± SEM of 12 determinations from three separate experiments. Means marked by different letters differ statistically at P b 0.05.
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Reagents Trans-Resveratrol (obtained via extraction from plant Bushy Knotweed), D-glucose, phorbol 12-myristate 13-acetate (PMA), mono-methyl succinate, acetylcholine chloride, cyclosomatostatin, bovine serum albumin (fatty acid free) and all reagents used to prepare Hanks' solution and Krebs–Ringer buffer and to determine blood glucose were obtained from Sigma. Dimethyl sulfoxide was from ICN Biomedicals Inc., whereas collagenase P (from Clostridium histolyticum) was purchased from Roche Diagnostics. The composition of Hanks' solution used for islet isolation was the following (in mM): NaCl 137, KCl 5.36, MgSO4 0.81, Na2HPO4 0.34, KH2PO4 0.44, CaCl2 1.26, NaHCO3 4.17. Krebs–Ringer buffer applied during incubations consisted of (in mM): NaCl 115, NaHCO3 24, KCl 5, MgCl2 1, CaCl2 1 and 0.5% (w/v) bovine serum albumin. Stock solutions of resveratrol, and PMA were prepared in dimethyl sulfoxide. The concentration of the solvent in the buffer with islets was less that 0.1%.
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Table 1 The effect of resveratrol on blood glucose, insulin and glucose/insulin ratio in normal rats Parameter
Control
Resveratrol 10 mg/kg body weight
Resveratrol 50 mg/kg body weight
Glucose (mM) Insulin (ng/ml) Glucose/insulin ratio (mM/ng/ml)
5.650 ± 0.262 1.345 ± 0.117 4.200 ± 0.299
6.067 ± 0.452 1.304 ± 0.132 4.652 ± 0.301
6.248 ± 0.320 1.027 ± 0.130⁎ 6.083 ± 0.351⁎
Resveratrol was dissolved in dimethyl sulfoxide:water mixture (4:6 v/v; 0.5 ml/ 100 g body weight) and was given intragastrically as a single dose 30 min before decapitation. Values represents the mean (n = 16) ± SEM. ⁎P b 0.05 vs. control and 10 mg resveratrol.
Means obtained in each group of rats (in vivo experiment) and from three separate experiments (in quadruplicates) in the case of insulin secretion studies were evaluated statistically using the analysis of variance and Duncan's multiple range test. Differences were considered significant at P b 0.05.
glucose. Acetylcholine increased hormone secretion by 130%. Under these stimulatory conditions, the influence of 1μM resveratrol on insulin secretion was negligible. However, despite the presence of acetylcholine, 10 and 100μM resveratrol still diminished secretion of insulin by 37% and 50%, respectively (Fig. 4). The direct activation of PKC by 500nM PMA in islets incubated with 6.7mM glucose resulted in a substantial increase (by 250%) in insulin secretion compared with islets exposed to glucose alone. The presence of 500nM PMA completely canceled the inhibitory effect of 1 and 10μM resveratrol, but resveratrol at the highest concentration (100μM) was still able to reduce (by 25%) the secretory response of β-cells (Fig. 4).
Results
In vivo experiment
In vitro experiments
Resveratrol administered to rats at the dose 10 or 50mg/kg body weight did not significantly affect blood glucose. The influence of resveratrol administered at low dose on blood insulin concentrations was also negligible. However, in rats receiving 50mg resveratrol/kg body weight a 24% decrease in insulinemia was found. Glucose/insulin ratio increased significantly (by 45%) only in animals treated with resveratrol at the dose 50mg/kg (Table 1).
Statistical analysis
Experiments with isolated pancreatic islets incubated for 90min with resveratrol revealed that basal release of insulin studied in the presence of 2.8mM glucose was unchanged by 1, 10 and 100μM resveratrol. However, insulin secretion induced by 6.7mM glucose was substantially reduced in islets exposed to resveratrol (1, 10 and 100μM). Similarly, maximal secretory response to 30mM glucose was clearly affected by the tested compound. In the presence of 1μM resveratrol insulin secretion tended to be reduced, but the effect was not statistically significant. Islets stimulated with 30mM glucose and exposed to 10 and 100μM resveratrol secreted significantly less insulin, by 20% and 30%, respectively, compared with untreated islets (Fig. 2). In depolarized islets, 10mM succinate enhanced insulin secretion by 380% compared with basal release. The insulinotropic action of this secretagogue was substantially blunted in the presence of 1, 10 and 100μM resveratrol by 24%, 30% and 25%, respectively (Fig. 3). The blockade of somatostatin receptors in islets stimulated with 6.7mM glucose resulted in increased secretion of insulin by 250% compared with glucose alone. Under these conditions, 1μM resveratrol failed to alter hormone secretion. However, islets exposed to 10 and 100μM resveratrol released less insulin by 18% and 40% than islets incubated without the tested compound (Fig. 3). Further studies demonstrated that in pancreatic islets 10μM acetylcholine potentiated the insulin-secretory response to 6.7mM
Discussion Results obtained in this study and in experiments performed previously (Szkudelski, 2006) clearly demonstrated that resveratrol incubated with rat pancreatic islets attenuated insulin secretion induced by glucose at physiological, supraphysiological and maximally effective concentrations, whereas basal hormone release remained unchanged. This indicates that the tested compound evinces its insulin-suppressive activity at a broad spectrum of stimulatory glucose concentrations ranging from 6.7 to 30 mM, but is completely ineffective at the non-stimulatory one. The lack of effects at the non-stimulatory concentration of glucose and a clearcut inhibition of secretion stimulated by succinate, a secretagogue metabolized exclusively in the mitochondria (Fahien and MacDonald, 2002), are in agreement with previous findings and provide further evidence confirming that the attenuation of insulin secretion caused by resveratrol is due to metabolic disturbances in β-cells. These disturbances involved diminished glucose oxidation, enhanced lactate production and reduced ATP levels. Moreover,
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glucose-induced hyperpolarization of mitochondrial membrane was attenuated in the presence of resveratrol (Szkudelski, 2007). The direct inhibitory effect of resveratrol on ATP synthase in the mitochondria isolated from rat brain and liver was also demonstrated (Zini et al., 1999; Zheng and Ramirez, 2000). On the other hand, Chen et al. (2007) recently showed that resveratrol increased insulin secretion from different β-cell insulinoma lines (MIN6, Hit-T15 and RIN-m5F). Such a distinction between whole islets and isolated β-cells could partially result from paracrine interactions within islets involving augmented secretion of somatostatin (Schatz and Kullek, 1980; Samols et al., 1986). The suppression of somatostatin action in islets canceled the inhibitory influence of 1 µM resveratrol. However, despite the blockade of somatostatin receptors, the insulin-suppressive effect of 10 and 100 µM resveratrol was still present. Since these observations were made in islets stimulated with 6.7 mM glucose, one can suppose that reduced secretion of insulin resulted from resveratrol-induced disturbances in metabolism of glucose. The most recent data provide evidence that resveratrol binds to SUR1, effectively displaces glibenclamide and blocks K+-ATP channels in mouse β-cells (Hambrock et al., 2007). The inhibitory influence of the tested compound on K+ current via these channels with a concomitant depolarization of plasma membrane was also shown in MIN6 cells (Chen et al., 2007). These effects should contribute to increased secretion of insulin. However, in experiments with freshly isolated rat pancreatic islets the opposite effects were found. Resveratrol reduced hormone secretion not only upon stimulation with glucose alone, but also in the presence of glibenclamide (Szkudelski, 2006) and under conditions when K+-ATP channels were bypassed (Szkudelski, 2007). Moreover, inhibition of succinate-induced insulin release was noted in islets exposed to diazoxide and high potassium to depolarize the plasma membrane. These secretory data allow the conclusion that interactions of resveratrol with K+-ATP channels are not involved in the attenuation of the endocrine function of rat β-cells, at least under experimental conditions applied in the present study and in the previous investigations (Szkudelski, 2006, 2007). Literature data indicate that resveratrol is able to inhibit PKC in different kinds of cells (Atten et al., 2001; Leiro et al., 2002; Woo et al., 2004). This effect is thought to arise from resveratrol's interaction with C1 domains of the enzyme (Slater et al., 2003). Activation of PKC in β-cells is involved in the mechanism of insulin secretion, especially at higher glucose concentrations (Sahai et al., 1992) and upon cholinergic stimulation (Persaud et al., 1989). In pancreatic islets incubated with 6.7 mM glucose and acetylcholine, 1 μM resveratrol failed to affect insulin secretion, but at concentrations 10 and 100 μM substantially aggravated this process. Furthermore, a direct activation of PKC by PMA completely suppressed the inhibitory action of 1 and 10 μM resveratrol on insulin secretion. PMA is usually present at concentration 100 nM to effectively activate PKC in β-cells. In the present study, the concentration of PMA was augmented to 500 nM since the interaction of resveratrol with PKC is competitive with respect to phorbol esters (Slater et al., 2003). However, despite high concentration of PMA, the inhibitory effect of resveratrol on insulin secretion was only partially suppressed. A similar effect was found when islets were exposed to 100 nM
PMA (Szkudelski, 2006). These results demonstrate that the inhibitory action of resveratrol on insulin secretion may be partially mitigated as a result of cholinergic stimulation. One can also suppose that inhibition of PKC activity is involved in the insulin-suppressive action of resveratrol in β-cells. In the light of previous data (Szkudelski, 2006, 2007) and the results of the in vitro experiments obtained in this study, it seems to be important to determine whether resveratrol may affect blood insulin levels. Literature data concerning resveratrol's effects on blood insulin are scanty. Su et al. (2006) revealed that in diabetic rats (a streptozotocin-nicotinamide model) resveratrol administered orally for a few days at a dose as low as 0.5 mg/kg body weight reduced blood insulin. Similarly, in mice with increased blood insulin as a result of a high-calorie diet, resveratrol (0.04% in the diet) also decreased insulinemia (Baur et al., 2006). In the present study, resveratrol administered perorally to normal rats as a single dose of 50 mg/kg body weight (but not at one fifth of that dose) diminished blood insulin. Simultaneously, glucose/insulin ratio increased. Since blood glucose was not significantly affected, one can suppose that the lowered insulinemia was due to the direct inhibitory effect of resveratrol on hormone secretion, as evidenced in vitro. In contrast to these outcomes, it was found that resveratrol administered orally (3 and 10 mg/kg body weight) (Chi et al., 2007) or intraperitoneally (3 mg/kg) (Chen et al., 2007) to normal rats increased blood insulin 20–90 min after administration. A similar effect was noted in diabetic rats with preserved ability to secrete insulin (Chi et al., 2007), but not in rats with type 1 diabetes (Chi et al., 2007; Chen et al., 2007). Data from the literature and the results of the present study indicate that doses of resveratrol necessary to induce changes in blood insulin are varied. However, these amounts are considerably below the toxic ones shown by Hebbar et al. (2005). In that study, rats were given 300, 1000 or 3000 mg resveratrol per kg body weight per day for four weeks; some harmful effects (nephrotoxicity, mild liver toxicity, moderate changes in enzyme activities and gene expression) were seen in rats receiving 1000 and 3000 mg resveratrol, but not in rats receiving 300 mg. In conclusion, results obtained in these studies demonstrated that resveratrol incubated with pancreatic islets isolated from normal rats did not affect basal insulin release, but clearly restricted physiological and maximal insulin-secretory response to glucose. The lack of influence on non-stimulated hormone release can be considered as a positive finding. It was also revealed that the inhibition of insulin secretion in islets exposed to resveratrol may be partially mitigated by acetylcholine, direct activation of PKC or blocking of somatostatin receptors. However, under these conditions resveratrol was still able to reduce insulin secretion, probably due to the inhibition of glucose metabolism. This is in accord to observations demonstrating that resveratrol does not disturb hormone secretion when this process is induced without metabolic events (Szkudelski, 2007). The obtained results strongly support the previous data that resveratrol exerts a clearcut insulin-suppressive action in rat pancreatic islets. Moreover, the experiment in vivo demonstrated that resveratrol is able to diminish blood insulin in normal rats provided that an appropriately large amount of this compound is administered. However, further studies are required to clarify resveratrol's
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