Nitrochalcones: Potential in vivo insulin secretagogues

Biochimie 91 (2009) 1493–1498

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Research paper

Nitrochalcones: Potential in vivo insulin secretagogues Rosangela Guollo Damazio a, Ana Paula Zanatta a, Luisa Helena Cazarolli a, Alessandra Mascarello b, Louise Domeneghini Chiaradia b, Ricardo Jose´ Nunes b, Rosendo Augusto Yunes b, Fa´tima Regina Mena Barreto Silva a, * a

´rio, Bairro Trindade, Cx. Postal 5069, ´gicas, Campus Universita Universidade Federal de Santa Catarina, Departamento de Bioquı´mica, Centro de Cieˆncias Biolo ´ polis, SC, Brazil CEP: 88040-970, Floriano b ´ticas, Campus Universita ´rio, Bairro Trindade, Universidade Federal de Santa Catarina, Departamento de Quı´mica, Centro de Cieˆncias Fı´sicas e Matema ´polis, SC, Brazil CEP: 88040-900, Floriano

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 June 2009 Accepted 1 September 2009 Available online 9 September 2009

In this study, the in vivo and in vitro anti-hyperglycemic activity of chalcone derivatives of 3,4-methylenedioxy, with a substituent electron-acceptor nitro group in the A or B ring, was investigated. As expected, the second generation sulfonylurea glipizide stimulated insulin secretion and reduced glycemia over the study period. Also, it was demonstrated for the first time that chalcones are able to increase insulin secretion and this event was coincident with serum glucose-lowering in the oral glucose tolerance test. Additionally, the chalcones studied had a similar effect on insulin secretion and serum glucose-lowering as glipizide. The effect of chalcones in terms of inducing insulin secretion was greater than that of glipizide after 30 min. Moreover, chalcones were not able to stimulate glucose uptake in soleus muscle, either in the presence of insulin or in the absence of this hormone. In addition, the oral treatment with chalcones did not alter glycemia in diabetic rats. These reports indicate that the effect of chalcones on serum glucose-lowering in hyperglycemic-normal rats is mainly a consequence of insulin secretion, highlighting these chalcones as novel compounds with strong anti-hyperglycemic properties. Ó 2009 Elsevier Masson SAS. All rights reserved.

Keywords: Chalcones Insulin secretion Hyperglycemia Diabetes

1. Introduction Maintenance of glucose homeostasis is achieved by the hormonal regulation of glucose uptake and endogenous glucose production, primarily by muscle and liver, respectively [1]. Insulin is the most important hormone in the regulation of blood glucose concentrations and energy metabolism [2,3]. On the other hand, glucose concentration deregulation is generated by a combination of impaired pancreatic insulin secretion, unsuppressed hepatic glucose production, and reduced glucose uptake by muscle and liver resulting in hyperglycemia [4,5]. Insulin is secreted into the blood stream by b-cells of the endocrine pancreas and glucose is the main insulin secretagogue [1]. Initially, glucose enters b-cells through the high capacity glucose transporter type 2 (GLUT 2) and is phosphorylated by glucokinase. The generation of ATP from glycolysis increases the intracellular ATP/ADP ratio. ATP binds ATP-dependent Kþ channels to the membrane of b-cells closing these channels and depolarizing these cells. The depolarization activates voltage-sensitive calcium

* Corresponding author. Tel.: þ55 48 3721 69 12; fax: þ55 48 37219672. E-mail address: [email protected] (F.R.M.B. Silva). 0300-9084/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2009.09.002

channels causing a calcium influx triggering insulin secretion [6,7]. Insulin secretion from pancreatic b-cells reflects the magnitude of fasting and postprandial blood glucose concentrations. Insulin secretion occurs in two distinct phases: an initial rapid release of insulin (Phase 1), when the glucose concentration increases after a meal, followed by a sustained increase in circulating insulin concentrations (Phase 2), according to glucose levels [8,9]. There are basically five classes of pharmacological agents with different mechanisms of action commonly used to target hyperglycemia, for example, meglitinides, biguanides, thiazolidinediones, alpha-glucosidase inhibitors and sulfonylureas [10]. Sulfonylureas (for example, glipizide) promote insulin secretion through binding to the sulfonylurea receptor (SUR) at the surface of b cells. The sulfonylurea receptor is intimately involved with subunits of an adenosine triphosphate-sensitive potassium channel (kir6.2). The binding of a sulfonylurea to the sulfonylurea receptor–kir6.2 complex results in closure of the potassium channels and inhibition of the efflux of potassium ions, promoting cell membrane depolarization, calcium influx and insulin secretion [11]. Chalcones (Fig. 1), compounds naturally found in plants or of synthetic origin, are known to exhibit several biological activities and have been involved in glucose metabolism [12–16]. It has been shown that chalcones isolated from plants have insulin-like

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O 2

2' 1'

3'

1

B

A 4'

6' 5'

3 4

6 5

Fig. 1. Basic structure of chalcones.

activities and improve the glucose uptake in adipocytes [17]. Also, it has been demonstrated that chalcones stimulate glucose uptake and potentiate insulin-stimulated glucose uptake in adipocytes [18]. Furthermore, chalcones derivatives from aryloxypropanolamines have shown potential anti-hyperglycemic effect when administered in hyperglycemic rats [19]. Taking into account the recent promissory effect of chalcones on serum glucose levels in hyperglycemic rats [20], the present study was carried out to investigate the role of 3,4-methylenedioxy substituted chalcones with an electronacceptor nitro group in insulin secretion, as well as the mechanism of action of these specific compounds in insulin target tissues. 2. Material and methods 2.1. Materials Glipizide and alloxan were purchased from Sigma Chemical CompanyÒ (St. Louis, MO, USA). Crystalline human insulin (100 IU/m; batch 20030H; BiohulinÒ – Biobra´s S.A, Montes Claros, MG, Brazil) was purchased from a commercial source. [U-14C]-2-deoxy-D-glucose ([14C]-DG), specific activity 10.6 GBq/mmol, and biodegradable liquid scintillation were obtained from Perkin Elmer Life and Analytical Sciences (Boston, MA). Salts and solvents were purchased from MerckÒ AG (Darmstadtm/Germany). 2.2. Chalcone synthesis Reagents used were obtained commercially (MerckÒ, Sigma– AldrichÒ). All chalcones were prepared by magnetic agitation of benzaldehyde and acetophenone, in methanol and KOH (50% v/v), at room temperature for 24 h [21]. Distilled water and 10% hydrochloric acid were added to the reaction for total precipitation of the compounds, which were then obtained by vacuum filtration and later recrystallized in dichloromethane and hexane. The structures were identified by their melting points (m.p.), infrared spectroscopy (IR), 1H and 13C nuclear magnetic resonance spectroscopy (NMR) and elementary analyses. 2.3. Experimental animals Male Wistar rats weighing 180–210 g were used. The rats were housed in plastic cages in an air-conditioned animal room and fed on pellets (Nuvital, Nuvilab CR1, Curitiba, PR, Brazil), with free access to tap water. Room temperature was controlled at 21  C with a 12 h light: 12 h dark cycle. Animals described as fasted had been deprived of food for 16 h but allowed free access to water. All the animals were monitored carefully and maintained in accordance with the ethical recommendations of the Brazilian Veterinary Medicine Council and the Brazilian College of Animal Experimentation (CEUA protocol PP00117/UFSC).

2.3.2. Study on effect of chalcones and glipizide on glucose tolerance curve Fasted normal rats were divided into different groups of eight rats. Group I, hyperglycemic control rats that received glucose (4 g/ kg; 8.9 M); Group II, treated hyperglycemic rats that received vehicle (corn oil): Group III, treated hyperglycemic rats that received different chalcones (1, 2, 3 and 4) (10 mg/kg); Group IV, treated hyperglycemic rats that received the oral antidiabetic drug glipizide (10 mg/kg). For all oral treatments, 0.5 ml of each substance was given by gavage and the serum glucose levels were measured immediately prior to, and at 15, 30, 60 and 180 min after treatment. After centrifugation, serum samples were used to determine serum glucose levels or for the insulin measurement. 2.3.3. Insulin serum measurements The insulin levels were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. The range of values detected by this assay was: 0.2–10 ng/ml. The intra- and inter-assay coefficients of variation (CV) for insulin were 3.22 and 6.95, respectively, with a sensitivity of 0.2 ng/ml. Analyses of area under curve (AUC) obtained during oral glucose tolerance curve (from zero to 60 min), were used to calculate insulinogenic index (Dins/Dgluc). All insulin levels were estimated by means of colorimetric measurements at 450 nm with an ELISA plate reader (Organon Teknika, Roseland, NJ, USA) by interpolation from a standard curve. Samples were analyzed in duplicate and results were expressed as ng of insulin serum ml1. 2.3.4. Studies on 14C-glucose uptake in rat soleus muscle For the [U-14C]-2-deoxy-D-glucose uptake (14C-DG) experiments, soleus muscles from normal rats were used. Slices of soleus muscle were distributed (alternately left and right) between basal and treated groups. The muscles were dissected, weighed, and preincubated and incubated at 37  C in Krebs Ringer-bicarbonate (KRb) buffer with a composition of 122 mM NaCl, 3 mM KCl, 1.2 mM MgSO4, 1.3 mM CaCl2, 0.4 mM KH2PO4, and 25 mM NaHCO3 and bubbled with O2/CO2 (95%:5%, v/v) up to pH 7.4. Chalcone 5 (104, 107, 109 and 1011 M), chalcone 6 (104, 107, 109 M) and insulin (108 M) were added to the pre-incubation (30 min) and incubation medium (60 min). The 14C-DG (0.1 mCi/ml) was added to each sample during the incubation period. After incubation, the muscles were transferred to screw cap tubes containing 1 ml of distilled water. These were frozen at 20  C in a freezer and boiled afterward for 10 min; 25 ml aliquots of tissue and external medium were placed in liquid scintillation in a LKB rack, beta liquid scintillation spectrometer (model 1215; EG and G-Wallac, Turku, Finland), for the radioactivity measurements. The results were expressed as the tissue/medium (T/M) ratio: cpm/ml tissue fluid per cpm/ml incubation medium [23]. 2.3.5. Study on effect of chalcones on serum glucose levels in diabetic rats Diabetes was induced by a single intravenous injection of alloxan monohydrate 5% (w/v) in a saline solution at a dose of 50 mg/kg body weight. Blood samples were collected 3 days later and glucose levels were determined to confirm the development of diabetes [24]. Fasted diabetic rats received chalcone 5 or chalcone 6 (10 mg/kg) by oral gavage. The serum glucose was measured immediately prior to, and at 1, 2 and 3 h following the treatment. 2.4. Data and statistical analysis

2.3.1. Determination of serum glucose levels Blood samples from the tail vein were collected, centrifuged and the serum was used to determine the glycemia by the glucose oxidase method [22].

Data were expressed as mean  S.E.M. One-way analysis of variance (ANOVA) followed by the Bonferroni post-test or unpaired Student’s t-test were used to determine the significant difference

R.G. Damazio et al. / Biochimie 91 (2009) 1493–1498

between groups. Differences were considered to be significant at P  0.05.

3.1. Acute effect of chalcone and glipizide on insulin secretion Although a broad range of biological activities have been described for chalcones, the action of chalcones on insulin secretion has not been reported. Based on the oral glucose tolerance curve of previously published studies, the specific chalcone 5 [20] and a chalcone (compound 1) from the present work were selected to study the potential effect on insulin secretion after in vivo treatment (Fig. 2A). Serum insulin levels were determined in fasted rats after an oral glucose loading at three selected times. Insulin secretion is subject to tight control by glucose. Glucose induced-insulin secretion was increased 128% at 15 min in hyperglycemic rats when compared with the euglycemic group, returning to the basal levels after 30 min (Fig. 2B). The rapid increase in insulin levels observed at 15 min after the oral glucose loading, in this model, is consistent with the profile of glucose induced-insulin secretion and is in agreement with the results of studies by Frangioudakis [25]. We observed that rats overloaded with glucose plus chalcones or glipizide have higher serum insulin levels than hyperglycemic rats. As expected a second generation sulfonylurea, glipizide, stimulated the insulin secretion at 15, 30 and 60 min by around 125, 83 and 130%, respectively, when compared to the hyperglycemic control group (Fig. 3). These results are in agreement with the literature concerning the effect of glipizide on insulin secretion in glucose tolerance tests [26]. Specifically, for the treatments with chalcones 1 and 5 the insulin secretion was significantly increased and the glucose effect on insulin levels was potentiated. Moreover, the stimulatory effect of chalcones in insulin secretion was noteworthy, since the maximum effect was at 30 min whereas for

A

O

O O O

O2N

Chalcone 5

O

Chalcone 1

B 4.0 Serum insulin (ng/ml)

NO2

O

Euglycemic Group Hyperglycemic Control

***

3.5 3.0

Hyperglycemic + Chalcone 5 Hyperglycemic + Chalcone 1

2.5 2.0

**

1.5

#

1.0

*** *

0.5 0

15

30 Time (min)

60

Fig. 2. (A) Structure of chalcone 5, (2E)-3-(1,3-benzodioxol-5-yl)-1-(40 -nitrophenyl)2-propen-1-one, and chalcone 1, (2E)-1-(1,3-benzodioxol-5-yl)-3-(30 -nitrophenyl)-2propen-1-one; (B) Effect of chalcones 5 and 1 (10 mg/kg) on serum insulin levels in hyperglycemic rats. Values are expressed as mean  SEM; n ¼ 8. Significant at # P  0.01 in relation to euglycemic group; Significant at *P  0.05, **P  0.01 and ***P  0.001 in relation to hyperglycemic control.

4.0 Serum insulin (ng/ml)

3. Results and discussion

1495

3.5

Euglycemic Group

3.0

Hyperglycemic Control

***

2.5 2.0

Hyperglycemic + Glipizide

*

1.5

*

#

1.0 0.5 0

15

30 Time (min)

60

Fig. 3. Effect of glipizide (10 mg/kg) on serum insulin levels in hyperglycemic rats. Values are expressed as mean  SEM; n ¼ 8. Significant at #P  0.01 in relation to euglycemic group; Significant at *P  0.05 and ***P  0.001 in relation to hyperglycemic control.

glipizide, an oral antidiabetic drug, it was observed at 15 min. In percentage terms, chalcone 1 stimulated insulin secretion by around 325% and chalcone 5 by around 265%, when compared with the respective time-course of the hyperglycemic control group. The treatment with chalcones 1 and 5 increase the insulinogenic index around 3 fold compared with the hyperglycemic control group (hyperglycemic control ¼ 0.29 ng/mg; chalcone 1 ¼ 1.04 ng/mg and chalcone 5 ¼ 0.96 ng/mg). In addition, the treatment with chalcone 1 and 5 did not alter the basal insulin secretion (data not shown). These results indicate, for the first time, the powerful effect of chalcones on insulin secretion. 3.2. Effect of chalcones and glipizide on serum glucose levels in hyperglycemic rats It has been recently demonstrated that chalcone derivatives from 3,4-methylenedioxybenzaldehyde, with an electron-acceptor nitro group at position 30 or 40 , were able to decrease glycemia in hyperglycemic-normal rats in oral glucose tolerance tests [20]. Thus, in this study, the effect of an electron-acceptor nitro group in 3,4-methylenedioxy chalcones on serum glucose levels in hyperglycemic-normal rats was examined. In the hyperglycemic control group, rats loaded with glucose by oral gavage reached a brief glycemia peak at 15 and 30 min, with glycemia returning to baseline levels at 180 min. In all experiments, the vehicle (corn oil) was not able to modify the hyperglycemic profile (data not shown). This study demonstrates that only chalcone 1 (Fig. 4A), with a nitro group at position 3, showed significant and acute anti-hyperglycemic activity in short-term treatment. The treatment with chalcone 1 decreased serum glucose levels in the oral glucose tolerance tests by 22, 31 and 14% at 15, 30 and 60 min, respectively, when compared to the hyperglycemic control group (Fig. 4B). On the other hand, chalcone 2 (Fig. 4A), structurally similar to chalcone 1, but with a nitro group at position 4, was not able to produce any anti-hyperglycemic effect (Fig. 4B). These results suggest that the steric conformation of the compounds is responsible for the anti-hyperglycemic activity of chalcone 1. However, Alberton [20] reported that chalcones with the presence of a nitro group at position 30 or 40 presented significant effects on serum glucose levels in hyperglycemic-normal rats. Also, chalcone 3 with a 3,4-methylenedioxy group in both rings (Fig. 5A), did not modify the glycemia when compared to the respective hyperglycemic control group (Fig. 5B). These results are

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R.G. Damazio et al. / Biochimie 91 (2009) 1493–1498 O

O

NO2

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O O

O

NO 2

Chalcone 1

Chalcone 2

Serum glucose level (mg/dL)

B 230

Hyperglicemic Control Hyperglycemic + Chalcone 1 10 mg/kg Hyperglycemic + Chalcone 2 10 mg/kg

Serum glucose level (mg/dL)

A

180

Hyperglycemic Control

230

Hyperglycemic + Glipizide 10 mg/kg

180

**

*** ***

130

**

80

******

0

15

30

60

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Time (min)

**

Fig. 6. Effect of glipizide on oral glucose tolerance curve. Values are expressed as mean  S.E.M; n ¼ 8. Significant at **P  0.01 and ***P  0.001 in relation to hyperglycemic control.

130

80 0 15 30

60

180 Time (min)

Fig. 4. (A) Structure of chalcone 1, (2E)-1-(1,3-benzodioxol-5-yl)-3-(30 -nitrophenyl)2-propen-1-one, and chalcone 2, (2E)-1-(1,3-benzodioxol-5-yl)-3-(40 -nitrophenyl)-2propen-1-one; (B) Effect of chalcones 1 and 2 on oral glucose tolerance curve. Values are expressed as mean  S.E.M; n ¼ 8. Significant at **P  0.01 and ***P  0.001 in relation to hyperglycemic control.

in agreement with that reported for chalcone (2E)-3-(1,3-benzodioxol-5-yl)-1-phenyl-2-propen-1-one with a 3,4-methylenedioxy group in the B ring (without substitution in the A ring) [20], reinforcing the importance of the presence of a nitro substituent in the rings for the chalcone biological activity. In order to clarify the importance of the presence of a nitro substituent at position 30 or 40 in the A ring, the effect of chalcone 4 with the electron-acceptor nitro group at position 20 in the same ring was studied (Fig. 5B). As expected, this chalcone was not able to modify the profile of the

A

glucose tolerance curve, verifying the importance of the substitution at position 30 or 40 in order to obtain the anti-hyperglycemic effect [20]. In order to compare the effect of chalcones with one of the drugs currently used as an antidiabetic, we assayed glipizide using this approach. Glipizide was able to reduce the glycemia significantly by 13, 26, 26, and 15% at 15, 30, 60 and 180 min, respectively, when compared with the hyperglycemic control (Fig. 6). This efficient and acute effect of glipizide reflects its efficacy in stimulating insulin secretion and consequently reducing serum glucose levels. Therefore, as chalcones exhibit a similar efficacy profile when compared with glipizide in oral glucose tolerance tests, these results indicate that chalcones represent potential insulin secretagogues.

3.3. Effect of chalcones on

14

C-glucose uptake in rat soleus muscle

It has been reported that both chalcones analogues and chalcones isolated from plants stimulate glucose uptake in 3T3-L1 NO2

O

O

O

O

O

O

O

O

Chalcone 3

Chalcone 4

Serum glucose level (mg/dL)

B

Hyperglycemic control 230

Hyperglycemic + Chalcone 3 10 mg/kg Hyperglycemic + Chalcone 4 10 mg/kg

180

130

80 0

15 30

60

180

Time (min) Fig. 5. (A) Structure of chalcone 3, (2E)-1,3-bis(1,3-benzodioxol-5-yl)- 2-propen-1-one, and chalcone 4, (2E)-3-(1,3-benzodioxol-5-yl)-1-(20 -nitrophenyl)-2-propen-1-one; (B) Effect of chalcones 3 and 4 on oral glucose tolerance curve. Values are expressed as mean  S.E.M; n ¼ 8.

14

1497

400

1.5

Serum glucose level (mg/dL)

C-DG uptake in soleus muscle (T/M)

R.G. Damazio et al. / Biochimie 91 (2009) 1493–1498

Control -4

[10 M] -7

1.0

[10 M] [10-9 M] [10-11 M]

0.5

0.0

Diabetic + Chalcone 5 10 mg/kg Diabetic + Chalcone 6 10 mg/kg

375 350 325 300 275 0

Chalcone 5 Fig. 7. Effect of chalcone 5 on 14C-glucose uptake in soleus muscle. Values are expressed as mean  S.E.M; n ¼ 4 in duplicate for each group.

adipocytes via a pathway involving PI3K and prevent the progression of diabetes [17,18]. However, there have been no reports on the effect of chalcones on glucose uptake in muscle cells. To assess the per se effect of chalcones on 14C-DG uptake and the synergic/additive insulin stimulatory effect on 14C-DG uptake in the soleus muscle, we used the chalcones with an electron-acceptor nitro group previously confirmed as showing anti-hyperglycemic activity [20]. Considering the dose-response curve relating to the effect of chalcones on glucose uptake in adipocytes, we obtained a doseresponse curve for chalcone 5 (104, 107, 109 and 1011 M) and chalcone 6 (104,107 and 109 M, without and with 108 M insulin). As expected, insulinstimulated significantly the 14C-DG uptake when compared to the control group and no stimulatory effect per se and/or synergic/additive effect were observed for these chalcones (Figs. 7 and 8). Nevertheless, in vivo, these chalcones improve the oral glucose tolerance curve [20]. In particular, chalcone 5 was effective in increase the serum insulin levels but was not able to influence glucose uptake in an insulin target tissue, indicating that this chalcone represents a potential insulin secretagogue. 3.4. Effect of chalcone on serum glucose levels in diabetic rats It has been demonstrated that chalcones with 3,4-methylenedioxy group located in the B ring are able to reduce the glycemia

A

O O2N

1

2 Time (h)

3

Fig. 9. Effect of chalcone 5 and chalcone 6 on serum glucose level in diabetic rats. Values are expressed as mean  S.E.M; n ¼ 8.

in streptozotocin-induced diabetic rats [19]. Also, chalcone from Angelica keiskei suppressed the elevation of blood glucose levels in genetically diabetic mice after chronic treatment [17]. Additionally, the anti-hyperglycemic activity of chalcones 5 and 6 in hyperglycemic-normal rats has been demonstrated [20]. Thus, we decided to verify the influence of these two chalcones on glycemia in alloxan-induced diabetic rats. As expected, the glycemia of alloxaninduced diabetic rats was not changed after an acute treatment with chalcone 5 and 6, respectively (Fig. 9). In this experimental model, the diabetic rats are unable to produce insulin, which shows that chalcones are effective in stimulating insulin secretion only in the presence of functional b cells. 4. Conclusions The results reported herein indicate that the effect of the tested compounds on serum glucose-lowering in hyperglycemic-normal rats is mainly a consequence of insulin secretion, verifying that these chalcones represent novel compounds with strong antihyperglycemic characteristics. Also, it reinforces that the position of the electron-acceptor nitro group in the chalcones, as well as the presence of the 3,4-methylenedioxy group in one of the rings, is essential for the biological activity to occur.

O

Acknowledgements O

14

C-DG uptake in soleus muscle (T/M)

Chalcone 6

B 2.0

Control

*** *** 1.5

[10 -4 M] [10 -7 M] [10 -9 M]

1.0

Chalcone 6 [10 -4 M] + Insulin [10 -8 M]

0.5

This study was supported by grants and fellowships from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico-Brasil (CNPq) and PIBIC-CNPq/Universidade Federal de Santa Catarina Program. Coordenaça˜o de Pessoal de Nı´vel Superior (CAPESPGFAR), and Fundaça˜o de Amparo a` Pesquisa do Estado de Santa Catarina (FAPESC). R. G. D. and L. H. C. are registered on the PGFARUFSC. A.M. and L. D. C. are registered on the PGQMC-UFSC. The author expresses their appreciation to Dr. Siobhan Wiese for assistance with the English correction of the manuscript. The authors express their appreciation to Dr. Danilo Wilhelm Filho and to the laboratory of Dr. Taˆnia Silvia Fro¨de for experimental support.

Insulin [10 -8 M]

References

0.0 Chalcone 6 (2E)-3-(1,3-benzodioxol-5-yl)-1-(30 -nitrophenyl)-2-

Fig. 8. (A) Structure of chalcone 6, propen-1-one; (B) Effect of chalcone 6 and insulin on 14C-glucose uptake in soleus muscle. Values are expressed as mean  S.E.M; n ¼ 4 in duplicate for each group. Significant at ***P  0.001 in relation to control group.

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