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Current Topics in Medicinal Chemistry, 2012, 12, 2049-2058 2049

Acylhydrazones Contribute to Serum Glucose Homeostasis Through Dual Physiological Targets

Marisa Jádna Silva Frederico1, Allisson Jhonatan Gomes Castro

1, Alessandra Mascarello

2,

Camila Pires Mendes1, Virgínia Dermachi Kappel

1, Taisa Regina Stumpf

2, Paulo Cesar Leal

2,

Ricardo José Nunes2, Rosendo Augusto Yunes

2 and

Fátima Regina Mena Barreto Silva

1,*

1Universidade Federal de Santa Catarina, Departamento de Bioquímica, Centro de Ciências Biológicas, Campus Uni-versitário, Bairro Trindade, Cx. Postal 5069, CEP: 88040-970, Florianópolis, SC, Brazil; 2Universidade Federal de Santa Catarina, Departamento de Química, Centro de Ciências Físicas e Matemáticas, Campus Universitário, Bairro Trindade, CEP: 88040-900, Florianópolis, SC, Brazil

Abstract: In this study the in vivo and in vitro antidiabetic effects of four acylhydrazone derivatives were investigated in

rats. The secretagogue action, oral glucose tolerance, insulinogenic index and mechanism of action of these acylhydra-

zones in relation to calcium uptake in pancreatic islets were studied. Also, the insulinomimetic effect on glycemia in dia-

betic rats was verified. Of the acylhydrazones studied, 1 and 4 were able to increase glucose tolerance in an acute time-

course. A powerful secretagogue effect was exhibited by 1 and glibenclamide with an insulinogenic index around 3.9 and

1.3-fold higher than that of the hyperglycemic group, respectively. Moreover, an acute and dose-dependent effect of

glibenclamide and 1 on calcium uptake in pancreatic islets was observed. The rapid stimulatory effect of 1 on calcium up-

take seems to be mediated, at least in part, by ATP-dependent K+ channels (K

+-ATP) since the stimulatory effect of 1 was

similar to that observed for glibenclamide but was not potentiated by sulphonylurea. Furthermore, both extracellular and

calcium from stocks mediate the signal transduction of stimulatory effect of 1 on calcium uptake which may contribute to

insulin secretion. In addition, the insulinomimetic effect of 1 was evidenced through the level of serum glucose lowering

in alloxan-induced diabetic rats. Also, 1 induced a significant increase in glycogen content in vivo and glucose uptake in

soleus muscle in vitro. The results of this study indicate dual physiological targets for the acylhydrazone 1, i.e., pancreatic

islets and skeletal muscle, as a result of insulin secretagogue and insulinomimetic action.

Keywords: Acylhydrazones, calcium, insulinomimetic, glucose, glycemia, pancreas, secretagogue, skeletal muscle.

INTRODUCTION

Diabetes mellitus (DM) is a global health problem and

one of the major causes of morbidity and mortality [1]. The

incidence of the disease is high worldwide and varies be-

tween populations due to genetic and susceptibility differ-

ences as well as other modifiable risk factors [2, 3]. While

the prevalence of DM has reached epidemic proportions,

most available treatments still focus on the symptoms of the

disease, rather than the underlying pathology itself. There are

basically two types of diabetes, type 1(T1DM) and type 2

(T2DM). T2DM accounts for the vast majority of cases of

diabetes, and is a heterogeneous disease marked by -cell

dysfunction [4, 5] combined with insulin resistance in target

tissues [4, 6, 7]. The pathogenesis of the disease involves

both genetic [8] and environmental factors, and is frequently

associated with obesity [6, 9, 10, 11]. T1DM is characterized

by the destruction of pancreatic -cells due to T-cell-

mediated auto-immune attack [12], which not yet fully un-

derstood.

*Address correspondence to this author at the Departamento de Bioquímica,

Centro de Ciências Biológicas, UFSC. Campus Universitário, Bairro Trin-

dade, Cx Postal 5069, CEP: 88040-970 - Florianópolis, Santa Catarina,

Brazil; Tel: +55 48 3721.6912; Fax: +55 48 3721.9672;

E-mail: mena@mbox1.ufsc.br

Types 1 and 2 diabetes have in common a deficit in -

cell mass. While the etiologies associated with T1DM and

T2DM differ, both forms are characterized by a relative defi-

cit in functional -cell mass such that the insulin require-

ments of the body are not met and glycemia is uncontrolled

[13]. Thus, studies that aim to investigate the mechanisms of

action of new compounds in insulin targets in order to high-

lights the diabetes treatments as well as improve glucose

tolerance, either through improving the insulin secretory

output of the remaining -cells (insulin secretagogues), as

well as improving the response to insulin in target tissues

(insulin sensitizers), or replacing the missing insulin (exoge-

nous insulinomimetic compounds), are of great importance

in relation to this pathogenesis.

Acylhydrazones structural skeleton is similar to the an-

tidiabetic compounds, chalcones, previously studied

[14,15,16]. Acylhydrazones compounds presents a wide

spectrum of biological properties including analgesic, anti-

inflammatory, antithrombotic, antinociceptive and antiplate-

let activity [17,18]. N-acylhydrazones have been shown to

produce a cardio inotropic effect and vasodilation of vascular

smooth muscle in rats [19]. Moreover, recently, the struc-

ture–activity relationships of 3,4,5-trimethoxy-hydrazones

has been described, these compounds showing potent anti-

cancer activity and the ability to provoke tubulin depolym-

1873-5294/12 $58.00+.00 © 2012 Bentham Science Publishers

2050 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 19 Frederico et al.

erization [20,21]. On the other hand, to the best of our

knowledge, the effect of acylhydrazones on diabetes was not

yet been reported. In this context, the aim of this study was

to determine the role of acylhydrazones on serum glucose

lowering and on insulin secretion as well as and the mecha-

nism of action associated with calcium uptake in pancreatic

islets. Insulomimetic effect of the acylhydrazone 1 in soleus

muscle was determined through glycogen content and glu-

cose uptake measurements. In addition, the acute toxicity of

acylhydrazones was investigated.

EXPERIMENTAL PROTOCOLS

Materials

Alloxan monohydrate [14], glibenclamide, diazoxide

[22], nifedipine [23], (1-[6-[((17 )-3-methoxyestra-

1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione) U-

73122 [21] and oyster glycogen type II were purchased from

Sigma Chemical Company (St. Louis, MO, USA). The io-

dine reagents (CaCl2 + I2 + KI) were purchased from

VETEC, Rio de Janeiro, Brazil. The commercial kit used to

determine the glycemia was produced by Analisa (Belo

Horizonte, MG, Brazil). The enzyme-linked immunosorbent

assay (ELISA) for quantitative determination of rat insulin

(catalogue no. EZRMI-13K) was purchased from Millipore

(St Charles, MO, USA). [45

Ca]CaCl2 (sp. act. 321 KBq/mg

Ca2+

), [U-14

C]-2-deoxy-D-glucose ([14

C]-DG), specific activ-

ity 10.6 GBq/mmol and biodegradable scintillation liquid

were obtained from Perkin Elmer Life and Analytical Sci-

ences (Boston, MA, USA). Salts and solvents were pur-

chased from Merck AG (Darmstadt. Germany).

For the in vivo experiments acylhydrazones were dis-

solved in a solution of 0.3% Tween 80. In all experiments,

the vehicle (solution of 0.3% Tween 80) alone was not able

to modify the results under any of the experimental condi-

tions, for the basal and treated groups (data not shown). For

the in vitro experiments 1 and 4 acylhydrazones were dis-

solved in dimethyl sulfoxide and added to both the control

and test media at final concentrations not exceeding 0.1%

(v/v). At this concentration, dimethyl sulfoxide failed to af-

fect islet function [24].

Experimental Animals

Male albino Wistar rats (180–210 g; 49 day-old) were

bred in an animal facility and housed in an air conditioned

room (approximately 21 ± 2 °C) with controlled lighting

(lights on from 06:00 to 18:00 h). Rats were maintained with

pelleted food (Nuvital, Nuvilab CR1, Curitiba, PR, Brazil),

and tap water was available ad libitum. For the in vivo and in vitro treatments rats were deprived of food for at least 16 h

but allowed free access to water. All the animals were moni-

tored and maintained in accordance with the ethical recom-

mendations of the Brazilian Veterinary-Medicine Council

and the Brazilian College of Animal Experimentation

(CEUA protocol PP00414/UFSC).

Studies on Effect of Acylhydrazones on Oral Glucose Tolerance

Hyperglycemic rats were divided into different groups of

four rats (see Tables and Figures), for the control and treated

animals. Control groups were randomly divided as follows:

rats which received vehicle (solution of 0.3% Tween 80);

and hyperglycemic control group of rats which received glu-

cose (4 g/kg; 8.9 M), p.o. gavage. Treated rats received the

acylhydrazones 1 (1, 10 and 100 mg/kg), 2, 3 and 4 (10

mg/kg) or glibenclamide (10 mg/kg) as a positive control

group, p.o. gavage. 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 glucose and insulin levels or lactate dehydro-

genase activity. Blood samples taken from the tail vein were

collected and centrifuged and the serum was used to deter-

mine the glycemia by the glucose oxidase method [25].

Insulin Serum Measurements

The insulin was measured applying the enzyme-linked

immunosorbent assay (ELISA) according to the manufac-

turer’s instructions. The range of values detected by this as-

say was 0.63 ng/mL to 4.3 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. All insulin

levels were estimated by means of colorimetric measure-

ments at 450 nm with an ELISA plate reader (Organon Tek-

nika, Roseland, NJ, USA) by interpolation from a standard

curve. Samples were analyzed in duplicate and results were

expressed as ng of insulin serum mL-1

. The incremental areas

under the response curves (AUCs) were calculated. The insu-

linogenic index (II) was calculated as the ratio between the

AUCinsulin and AUCglucose (from zero to 60 min) [14].

Pancreatic Islet Isolation

The rat pancreas was visualized by way of a central ab-

dominal incision. The bile duct was clamped at the tip of the

duodenum and cannulated at a point sufficiently proximal to

the liver. Krebs Ringer bicarbonate buffer with a composi-

tion of 122 mM NaCl, 3 mM KCl, 1.2 mM MgSO4, 1.3 mM

CaCl2, 0.4 mM KH2PO4, and 25 mM NaHCO3 supplemented

with 8 mM HEPES (KRb-HEPES) medium was introduced

slowly into the bile duct by syringe until the pancreas was

clearly distended. The pancreas was then gently removed and

then kept in Petri dishes with KRb-HEPES medium. The

pancreatic tissue was cut into small pieces (2 x 2 mm) and

incubated in plastic tubes in 1 mL of KRb-HEPES medium

supplemented with collagenase (3 mg). After incubation the

mixture was transferred to a conical tube (110 x 15 mm),

resuspended in 10 mL with collagenase free medium and

centrifuged at room temperature for 3 min at 45 x g in an

Excelsa Baby centrifuge (model 206), FANEM, São Paulo,

SP, Brazil. The supernatant was discarded and the sediment

resuspended in fresh KRb-HEPES medium. This washing

procedure was repeated five times; in the last two washings

the islets were allowed to settle without centrifugation. Ali-

quots (100 L) of the final sediment with the isolated islet

were transferred to eppendorff tubes with the incubation me-

dium KRb-HEPES [26,27].

Calcium Uptake Experiments

The isolated islets were pre-incubated for 60 min in a

Dubnoff metabolic incubator to equilibrate in KRb-HEPES

buffer containing 5 mM glucose and 0.1 Ci/mL45

Ca2+

at

Antihyperglycemic and Hypoglycemic Effects of Acylhydrazones Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 19 2051

37°C, pH 7.4 and gassed with O2:CO2(95:5; v/v). The islets

were incubated for 10 min in KRb-HEPES without (control)

or with the acylhydrazone 1 (0.2, 2 and 20 M). In some

experiments a channel antagonist, agonist or phospholipase

C (PLC) inhibitor were added during the last 15 min of incu-

bation before the treatment and maintained during the entire

incubation period (see figure legends). The following drugs

were used: nifedipine (0.1, 1 and 10 M), diazoxide (250

M) [28,29], glibenclamide (0.2, 2 and 20 M), U-73122 (1

M) [23]. The incubation was halted using the technique

reported by Batra and Sjogren [30] with modifications [28].

Cold buffer (1 mL with lanthanum chloride (10 mM) at 2°C

was added to the samples to stop calcium fluxes. The tubes

were centrifuged for 1 min at 45 x g. The supernatant was

preserved and the islets were washed twice in cold lantha-

num chloride solution. The islets were added to screw-cap

tubes containing 300 L of 0.5 M NaOH solution and boiled

at 100°C for 5 min. Aliquots of 50 L were taken from each

sample for the measurement of radioactivity in scintillation

liquid Optiphase Hisafe III (Wallac Oy, Turku, Finland) in

an LKB rack beta liquid scintillation spectrometer (model LS

6500; Multi-Purpose Scintillation Counter-Beckman Coulter,

Boston, USA) and 5 L aliquots were used for protein quan-

tification by the Lowry method [31].

Studies on Effect of Acylhydrazones on Serum Glucose Levels in Diabetic Rats

Diabetes was induced by a single intravenous injection of

5% (w/v) alloxan monohydrate 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 [14]. Fasted diabetic rats received

the acylhydrazone 1 (10 mg/kg) by oral gavage. The serum

glucose was measured immediately prior to and after 1, 2

and 3 h of treatment.

Skeletal Muscle and Liver Glycogen Measurements

Soleus muscle and liver were harvested from hypergly-

cemic rats, untreated and treated with the acylhydrazone 1

(10 mg/kg), and then used for the assay of glycogen content

immediately after 3 h of treatment. Glycogen was isolated

from these tissues as described by Krisman [32], with minor

modifications [33]. The tissues were weighed, homogenized

in 33% KOH and boiled at 100°C for 20 min, with occa-

sional stirring. After cooling, 96% ethanol was added to the

samples which were heated to boiling followed by cooling in

an ice bath to aid the precipitation of glycogen. The ho-

mogenates were centrifuged at 1300 x g for 15 min, the su-

pernatant was discarded and the pellets were neutralized with

saturated NH4Cl before being heated to 100°C for 5 min,

washed and resolubilized in water. Glycogen content was

determined by treatment with iodine reagent and the absor-

bance was measured at 460 nm. The results are expressed as

mg of glycogen/g of tissue.

Studies on Glucose Uptake in Rat Soleus Muscle

For the [U-14

C]-2-deoxy-D-glucose uptake experiments,

soleus muscles from normal fasted rats were used. Soleus

muscles were distributed (alternately left and right) between

basal and treated groups. The muscles were dissected and

then preincubated (30 min) and incubated (60 min) at 37°C

in KRb buffer with 3 mM glucose bubbled with O2/CO2

(95%:5%, v/v) until pH 7.4. The acylhydrazone 1 (20 M)

was added to the incubation medium (60 min) in the pres-

ence of 14

C-DG (0.1 Ci/mL). After incubation the muscles

were placed in screw cap tubes containing 500 L of a 0.5 M

NaOH solution and boiled at 100°C for 5 min. Aliquots of 25

L were taken from each sample for radioactivity measure-

ments in scintillation liquid Optiphase Hisafe III (Wallac Oy,

Turku, Finland) on an LKB rack beta liquid scintillation

spectrometer (model LS 6500; Multi-Purpose Scintillation

Counter-Beckman Coulter, Boston, USA) and 5 L aliquots

were used for protein quantification by the Lowry method

[31]. The results were expressed as the nmol glycosyl units/

mg protein [34], with modifications.

Lactate Dehydrogenase Assay (LDH Assay)

The in vivo toxicity was assessed by measuring the re-

lease of the cytosolic enzyme lactate dehydrogenase into

serum after 180 min of treatment with the acylhydrazone 1.

The LDH activity was measured applying a spectropho-

tometric method and the level of activity was calculated by

measuring the oxidation of NADH with the results being

expressed as % of control [35].

Data and Statistical Analysis

Data were expressed as mean ± S.E.M. One-way analysis

of variance (ANOVA) followed by the Bonferroni post-hoc test or unpaired Student’s t-test was used to determine the

significance of differences between groups. Differences were

considered to be significant at p 0.05.

Chemistry

The acylhydrazones were prepared by condensation of

3,4,5-trimethoxybenzohydrazide and the appropriate alde-

hyde, under reflux [36]. The 3,4,5-

trimethoxybenzohydrazide was prepared as previously de-

scribed [37]. Details on the synthesis of the compounds have

been previously published [15,16,21].

RESULTS AND DISCUSSION

Studies on Effect of Acylhydrazones on Oral Glucose

Tolerance

The results related to the effect of acylhydrazones and

glibenclamide on serum glucose levels in hyperglycemic rats

can be seen in Fig. (1) and (Table 1). Initially, a dose-

response curve on oral glucose tolerance for the acylhydra-

zone 1 (1, 10 and 100 mg/kg), was obtained. As expected, in

the hyperglycemic control group after starting the oral glu-

cose tolerance test, the serum glucose level was typically

increased when compared with zero time for the same group.

However, at a dose of 10 mg/kg the acylhydrazone 1

produced remarkable serum glucose lowering at 15, 30 and

60 min (28%, 22% and 19%, respectively) compared to the

hyperglycemic control group. As it can be seen at Fig. 1, the

oral hypoglycemic compound, glibenclamide, produced a

serum glucose lowering after 15, 30 and 60 min at dose of 10

mg/kg (23%, 32% and 32%) compared to the hyperglycemic

control group, respectively. This dose of glibenclamide is in

line with that reported by Sakamoto et al. [38] and León-

Reyes et al. [39], concerning the significant therapeutic

effect on glycemic control. On the other hand, at doses of 1

2052 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 19 Frederico et al.

and 100 mg/kg of the acylhydrazone 1 slightly reduced

glycemia (by around 14% and 12%) only after 30 min,

respectively [Fig. (1)]. Based on these results, the dose of 10

mg/kg was used for further studies with other

acylhydrazones.

Our previous studies identified the substituents at differ-

ent positions on the core structure of chalcones that deter-

mine their antihyperglycemic activity as well as the specific-

ity of action [14,40,41]. Taking in mind that the structural

skeleton of acylhydrazones is similar to that of chalcones,

some selected modifications to the core structure of acylhy-

drazones were made to assay them in vivo and in vitro

[Scheme 1].

Scheme 1. Structure of the acylhydrazones.

Based on the preliminary results for 1 (substituent 1-

naphthyl), we tested the structural analogue of 2 (substituent

2-naphthyl). As can be observed in (Table 1), the compound

2 produced serum glucose lowering of around 20% and 11%

at 15 and 30 min, respectively, when compared with

hyperglycemic control group. These data indicate that 2 was

less potent than compound 1, namely due to a change in the

conformation of the naphthyl group. Also, we analyzed the

influence of an electron donor group in the core of the

acylhydrazone. The oral administration of 3 (substituent p-

N(CH3)2) slightly reduced the serum glucose by 18% and

23% at 15 and 30 min, respectively [Table 1]. On the other

hand, the oral administration of an acylhydrazone with the

electron acceptor group 4 (substituent p-Br) showed signifi-

cant, acute and lasting serum glucose lowering, of around

25%, 1.8% and 16% at 15, 30 and 60 min, respectively.

Based on the results outlined herein the development of a

series of novel molecules is underway in order to improve

the biological activity.

This first goal was to demonstrate that the acylhydra-

zones 1 and 4 markedly reduce serum glucose levels and

then to determine the insulinogenic index, mechanism of

action and insulinomimetic effect for these compounds.

Effect of Acylhydrazone and Glibenclamide on Serum Insulin Measurements

(Table 2) shows a typical pattern of insulin secretion after

glucose overloading (4 g/kg) in hyperglycemic control

groups with an insulinogenic index (II) of 0.66. The acylhy-

drazones 1 and 4 exhibited high II values of around 2.5.

Also, the acylhydrazone 1 showed a potent insulin

secretagogue effect within 15 min after oral treatment. As it

was expected, the glibenclamide was effective to induce in-

sulin secretion exhibiting an II around 2. It is reported that

the most important pathway for quantitative insulin release

from islet interstitial is regulated by Ca2 +

ion [42-47]. In this

context, the mechanism of action of this efficient insulin

secretagogue and antihyperglycemic compound, acylhydra-

zone 1, was studied in calcium uptake in pancreatic islets.

Effect of Acylhydrazone on Ca2+

Uptake in Pancreatic Islets

After the equilibrium of calcium in the pancreatic islets

following 60 min of incubation with 45

Ca2+

(0.1 Ci/mL),

calcium measurements were carried out after 10 min in the

presence of glibenclamide or the acylhydrazone 1 (0.2, 2 and

20 M). (Table 3) shows that glibenclamide stimulated sig-

nificantly the calcium uptake at 20 M. Percentile, the dose

of 20 M of the acylhydrazone 1 showed a stimulatory effect

on calcium uptake around 3.0 fold (p < 0.001) greater than

that observed for the control group after 10 min. The rapid

calcium uptake is associated with the first phase of insulin

secretion which is sustained due to two functional pools de-

fined as the readily releasable pool and the immediately re-

leasable pool [48,49]. This is consistent with the literature

where it is demonstrated that an intracellular calcium in-

crease in –cells is essential for exocytose of insulin gran-

ules [50-54]. Therefore, based on the dose-response curve, in

all subsequent experiments 20 M of the acylhydrazone 1

and a 10 min incubation period were used, since 10 min is

related to the first phase of insulin release in –cells.

In the –cell, the closing of potassium channels leads to

the depolarization of the plasma membrane with subsequent

opening of voltage-dependent Ca2+

channels and acceleration

of Ca2+

uptake. This abrupt increase in the concentration of

cytosolic-free Ca2+

([Ca2+

]i) is mandatory for the triggering

of insulin secretion [53-57]. Thus, the mechanism of action

of the acylhydrazone 1 associated with the stimulatory effect

on 45

Ca2+

uptake was investigated in pancreatic islets. To

assess the involvement of potassium channels in the

stimulatory effect of the acylhydrazone 1 on calcium uptake

one antagonist and one agonist of potassium channels were

used, that is, glibenclamide (20 μM) and diazoxide (250 μM)

[22], respectively.

Compound 1 was able to increase significantly the

calcium uptake, by around 77% [Fig. (2A)]. Glibenclamide,

a blocker of K+-ATP channels, stimulated calcium uptake by

a factor of 1.5 when compared with the control. On the other

hand, when glibenclamide was preincubated before 1, a

significant decrease in the stimulatory effect of 1 on calcium

Antihyperglycemic and Hypoglycemic Effects of Acylhydrazones Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 19 2053

Fig. (1). Dose-response curve of acylhydrazone 1 administered by oral gavage on glucose tolerance. Each value represents mean ± S.E.M. of

four determinations. bP 0.01,

cp 0.001 compared to hyperglycemic control group.

Table 1. Effects of Acylhydrazones 1, 2, 3 and 4 (10 mg/kg) on Glucose Tolerance Curve.

Serum glucose levels (mg/dL) Time (min)

0 15 30 60 180

Hyperglycemic 102.29 ± 6.34 177.46 ± 5.95 190.15 ± 1.77 182.55 ± 6.95 141.15 ± 4.47

Glibenclamide 112.06 ± 5.61 140.49 ± 4.13c 135.24 ± 5.67

c 121.67 ± 5.15

c 111.45 ± 3.08

c

1 108.35 ± 1.39 126.76 ± 1.39c 148.08 ± 3.26

c 146.90 ± 4.46

c 145.34 ± 1.72

2 116.04 ± 2.53 142.39 ± 3.39c 169.48 ± 6.88

a 167.96 ± 11.06 132.08 ± 3.51

3 115.38 ± 3.38 146.08 ± 5.81c 145.24 ± 5.34

c 159.97 ± 4.15 145.20 ± 1.66

4 103.64 ± 2.32 131.98 ± 4.71c 154.48 ± 6.52

c 152.43 ± 8.33

c 131.44 ± 6.79

Each value represents mean ± S.E.M. of four determinations in triplicate. aP 0.05,

cp 0.001 compared to hyperglycemic control group.

Table 2. Effect of Acylhydrazones 1 and 4 and Glibenclamide (10 mg/kg) on Serum Insulin Secretion.

Group Serum insulin levels (ng/mL) Time (min)

0 15 30 60 II*

Hyperglycemic 0.09 ± 0.001 2.84 ± 0.002 0.75 ± 0.007 0.59 ± 0.004 0.66

Glibenclamide - 3.83 ± 0.430a 2.66 ± 0.001

b 2.46 ± 0.070

c 1.98

1 - 3.35 ± 0.001c 0.53 ± 0.009 0.20 ± 0.026 2.55

4 - 2.21 ± 0.550 0.94 ± 0.045 0.56 ± 0.095 2.53

Each value represents mean ± S.E.M. of four determinations in triplicate. aP 0.05,

bp 0.01, cp 0.001 compared to hyperglycemic control group. *Insulinogenic index.

0 15 30 60 18075

100

125

150

175

200

Acylhydrazone 1 (1 mg/kg)

Acylhydrazone 1 (10 mg/kg)

Hyperglycemic

c

Acylhydrazone 1 (100 mg/kg)

c c

c

b

b

Glibenclamide (10 mg/kg)

c

c

cc

Time (min)

Seru

m g

luco

se le

vel (

mg/

dL)

2054 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 19 Frederico et al.

Table 3. Dose-Response Curve of Glibenclamide and Acylhydrazone 1 on 45

Ca2+

Uptake in Pancreatic Islets.

Group 45

Ca2+

uptake (% of control)

0.2 M 2 M 20 M

Glibenclamide 132.14 ± 7.58 134.79 ± 7.51 156.72 ± 9.67b

1 136.50 ± 4.24 138.96 ± 5.61 301.05 ± 2.86c

Each value represents mean ± S.E.M. of four determinations in triplicate. bP 0.01

cp 0.001 compared to control group.

uptake was observed when compared with the 1 group alone.

It is believed that 1 may block K+-ATP channels or to

destabilize the binding of glibenclamide on K+-channel. It

has been reported that, in fact, sulfonylureas are considered

as partial antagonists of the K+-ATP channels, since that the

K+-ATP channel blockage is not complete [43]. It has been

described that the maximum binding of sulphonylureas on

K+-ATP channel is around 60-80%, observed from

sulphonylurea concentration-inhibition curve [58]. Thus,

tests were also carried out to verify the effect of 1 after

glibenclamide incubation. As can be observed in Fig. (2B), the stimulatory effect of 1 on calcium uptake was exactly the

same with or without glibenclamide. From these results, it

can be suggested that the stimulatory effect of 1 on calcium

uptake strongly involves K+ channel activity. These results

were corroborated by the use of diazoxide, a potassium

channel agonist that was able to decrease (23%) calcium

uptake induced by the acylhydrazone 1 [Fig. (2C)]. Consid-

ered overall, these results are in line with the literature

[22,49], in which it is demonstrated that diazoxide decreases

the efficacy of Ca2+

in insulin exocytosis. Our results

strongly suggest that the participation of potassium channels

plays a crucial role in the mechanism of action of 1 involved

in the stimulatory effect on calcium uptake in -cells.

It is well known that the depolarization of the plasma

membrane induced by sulfonylureas activates L-type

calcium channels in pancreatic islets [59,43,44]. Therefore,

the involvement of the L-type calcium channels and PLC in

the stimulatory effect of 1 in pancreatic islets was

investigated. In this approach, a dose-response curve for

nifedipine at 0.1, 1 and 10 M in relation to basal calcium

was obtained. Any alteration on basal calcium uptake was

observed at 1 and 10 M nifedipine (data not shown). So, 1

M was used to the further experiments. In Fig. (3A), it is

demonstrated that 1 μM nifedipine, after 15 min of incuba-

tion, did not alter the basal calcium cellular equilibrium.

However, it was found that the rapid 1-induced elevation of 45

Ca2+

uptake was prevented in the presence of the L-type

voltage-dependent calcium channel (VDCC) blocker nifedip-

ine.

Importantly, Ca2+

uptake can also be produced or en-

hanced by mechanisms that are independent of plasma mem-

brane depolarization induced by K+-ATP channel activity

[60,61]. Fig. (3B) showed that in the presence of a phosphol-

ipase C inhibitor (U-73122) the stimulatory effect on

calcium uptake generated by 1 was partially blocked. In

addition, other studies have indicated that the PLC plays a

significant role in maintaining the quantity of intracellular

Ca2+

, in particular, in the second phase of insulin secretion

[48, 62]. Our results also suggest the participation of calcium

from stocks in the mechanism of action of 1 in pancreatic

islets, an event involved in insulin secretion. These results

are in line with the rapid in vivo insulin secretion induced by

1 shown in (Table 2).

Studies on the Effect of Acylhydrazones on Serum Glu-

cose Levels in Diabetic Rats

Since alloxan causes a destruction of -cells, this is a

useful approach to study the role of compounds with

potential antidiabetic effect [63]. Fig. (4) shows that 1

significantly decreased glycemia by around 25% after 2 h of

treatment. Antidiabetic compounds can regulate glucose lev-

els by increasing glucose uptake, augmenting glycogen con-

tent and/or inhibiting the intestinal glucose absorption,

among other specific targets involved in glucose metabolism

[64]. Thus, in order to investigate the insulinomimetic role of

1, the acute effect of 1 on the glycogen content of muscle

and liver tissue in hyperglycemic rats was analyzed.

Effect of acylhydrazones on glycogen content

(Table 4) shows a significant increase in the muscle

glycogen content of around 2.2 and 2.5 fold following

treatments with 1 and 4 acylhydrazone, respectively. On the

other hand, neither 1 nor 4 were able to change the liver

glycogen content. These results indicate that 1 acts directly

on peripheral tissue (skeletal muscle). These particular re-

sults are interesting since, to the best of our knowledge, this

is the first time that an exogenous compound with this

chemical characteristic is reported to increase significantly

glycogen content in an insulin tissue target (skeletal muscle).

For this reason, since the 1 compound increased skeletal

muscle glycogen content as well as diminished glycemia in

alloxan-induced diabetic rats, the effect of 1 on glucose

uptake in soleus muscle was evaluated.

Effect of Acylhydrazone 1 on Glucose Uptake in Rat So-

leus Muscle

(Table 5) shows the in vitro effect of the acylhydrazone 1

(20 μM) on glucose uptake in the rat soleus muscle follow-

ing 60 min of incubation. As it can be observed, compound 1

stimulated 14

C-DG uptake to a remarkable extent when com-

pared to the control group. Overall, on considering the data

reported herein [Fig. (4), Table 4, Table 5], the

acylhydrazone 1 can be classified as an insulinomimetic

agent. Our results are in line with Kawabata et al. [65], who

reported that prenylated chalcones 4-hydroxiderricin and

xanthoangelol stimulate glucose uptake in L6 muscle cells,

contributing to glucose homeostasis.

Antihyperglycemic and Hypoglycemic Effects of Acylhydrazones Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 19 2055

Fig. (2). Involvement of K+-ATP channels in the stimulatory effect

of the acylhydrazone 1 on 45

Ca2+

uptake in pancreatic islets. (A),

glibenclamide (20 μM) (B), 1 (20 μM) and (C), Diazoxide (250

μM) were present in the last 15 min of pre-incubation and during

the incubation period. Pre-incubation time: 60 min; incubation time:

10 min. Means ± S.E.M. of 3 independent experiments. aP 0.05,

bp 0.01, cp 0.001 compared to control group.

# Compared to 1

group.

Fig. (3). Involvement of L-type VDCC and intracellular calcium

stocks in the stimulatory effect of acylhydrazone 1 on 45

Ca2+

uptake

in pancreatic islets. (A), Nifedipine (1 μM) (B), U-73122 (1 M)

were present in the last 15 min of pre-incubation and during the

incubation period. Pre-incubation time: 60 min; incubation time: 10

min. Mean ± S.E.M. of 3 independent experiments. p 0.001 com-

pared to control group. #

Compared to 1 group.

Effect of Acylhydrazones on LDH Activity

The tissue viability was analyzed through the LDH

activity, measuring the release of the cytosolic enzyme

lactate dehydrogenase into the serum by damaged tissue. The

oral gavage treatment with the acylhydrazones 1 and 4 for

180 min was not able to induce tissue damage as can be ob-

served in (Table 6). Also, in similar concentration range of

acylhydrazones low level of cytotoxicity was reported by

Tian et al. [66].

CONCLUSION

In a whole, the in vivo and in vitro effects of the acylhy-

drazone 1 show at least dual strategic targets involved in

glucose homeostasis, that is, the pancreas and skeletal mus-

cle. The highlighted mechanism of action described herein

A

0

15

30

45

Control1 20 μMGlibenclamide 20 μMGlibenclamide 20 μM + 1 20 μM

c

ac

a

pmol

45C

a2+/μ

g pr

otei

n

#

B

0

15

30

45

60

75

ControlGlibenclamide 20 μM1 20 μM1 20 μM + Glibenclamide 20 μM

b b

a

pmol

45C

a2+/μ

g pr

otei

n

C

0

15

30

45

Control1 20 μMDiazoxide 250 μMDiazoxide 250 μM + 1 20 μM

c

a

pmol

45C

a2+/μ

g pr

otei

n

#

A

0

15

30

45

Control1 20 μMNifedipine 1 μMNifedipine 1 μM + 1 20 μM

c

a

pmol

45C

a2+/μ

g pr

otei

n

c#

B

0

15

30

45

60

Control1 20 μMU-73122 1 μMU-73122 1 μM + 1 20μM

c

pmol

45C

a2+/μ

g pr

otei

nb#

2056 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 19 Frederico et al.

Fig. (4). Effect of acylhydrazone 1 on fasted serum glucose levels in diabetic Wistar rats, means ± SEM, n= 6, cp 0.001 compared to zero

time.

Table 4. Effect of Acylhydrazones 1 and 4 (10 mg/kg) on Glycogen Content in Rats.

Group

Hyperglycemic 1 4

Muscle 7.72 ± 0.40 16.81 ± 0.61c 18.76 ± 2.16

c

Liver 14.60 ± 1.27 11.71 ± 2.81 16.10 ± 1.23

Each value represents the mean ± S.E.M. of four determinations. cP 0.001 compared to hyperglycemic control group. Results expressed in mg/g of tissue.

strongly suggests both an insulin secretagogue and insuli-

nomimetic role for this compound. The effects of the acyl-

hydrazone 1 on serum glucose lowering and insulin secre-

tion, and its acute stimulatory action on calcium uptake in

pancreatic islets, glycogen accumulation and glucose uptake

in skeletal muscle on in vivo treatment, did not indicate cyto-

toxicity. Finally, the acylhydrazone 1 appears to represent a

potentially useful candidate for diabetes therapy in the near

future.

Table 5. Effect of 20 M Acylhydrazone 1 on Glucose Uptake

Glucose uptake (nmol glysosyl units/ mg protein)

Control 0.22 ± 0.02

1 0.77 ± 0.18b

Each value represents the mean ± S.E.M. of three determinations. bP 0.01, compared

to control group.

Table 6. Effect of Acylhydrazones 1 and 4 on Extracellular

LDH Activity in Serum

Group LDH % of Control Time (180 min)

1 115.2 ± 11.9

4 116.6 ± 5.8

1 and 4 (10 mg/kg). Each value represents the mean ± S.E.M. of four determinations.

CONFLICT OF INTEREST

The author(s) confirm that this article content has no con-

flicts of interest.

ACKNOWLEDGEMENTS

This study was supported by grants and fellowships from

the governmental agencies Conselho Nacional de Desenvol-

vimento Científico e Tecnológico-Brasil (CNPq), Coordena-

ção de Pessoal de Nível Superior (CAPES-PPG-Bioquímica)

and Fundação de Amparo à Pesquisa do Estado de Santa

Catarina (FAPESC). We also thank to Louise D. Chiaradia

for the support chemical. M. J. S. F is registered on the PPG-

Bioquímica. C. P. M. (CNPq fellowship).

ABBREVIATIONS

DM = Diabetes Mellitus

T1DM = Diabetes type1

T2DM = Diabetes type 2

U-73122 = 1-[6-[((17 )-3-methoxyestra-1,3,5[10]-

trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-

dione

II = Insulinogenic index

AUCs = Areas under the response curves

KRb-HEPES = Krebs Ringer bicarbonate buffer with

HEPES

LDH = Lactate dehydrogenase

0 1 2 3

100

250

400

550Acylhydrazone 1 (10 mg/kg)

c

Time (h)

Seru

m g

luco

se le

vel (

mg/

dL)

Antihyperglycemic and Hypoglycemic Effects of Acylhydrazones Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 19 2057

PLC = Phospholipase C

VDCC = L-type voltage-dependent calcium channel

K+-ATP = ATP-dependent K

+ channel

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Received: July 05, 2012 Accepted: October 11, 2012