ORIGINAL CONTRIBUTION

Protective effects of garlic extract on cardiac function, heart ratevariability, and cardiac mitochondria in obese insulin-resistantrats

Luerat Supakul • Hiranya Pintana •

Nattayaporn Apaijai • Siriporn Chattipakorn •

Krekwit Shinlapawittayatorn • Nipon Chattipakorn

Received: 17 July 2013 / Accepted: 3 October 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract

Purpose Garlic has been shown to exhibit antioxidant

effects and cardioprotective properties. However, the

effects of garlic extract on the heart in insulin resistance

induced by long-term high-fat-diet consumption are not

well defined. Therefore, we sought to determine the effects

of garlic extract in the obese insulin-resistant rats.

Methods Male Wistar rats (180–200 g) were divided into

two groups: normal-diet or high-fat-diet (n = 24/group)

fed for 12 weeks. Rats in each groups were divided into

three subgroups (n = 8 each): vehicle or garlic extract (250

or 500 mg/kg/day, respectively) treated for 28 days. At the

end of the treatment, the metabolic parameters, heart rate

variability (HRV), cardiac function, and cardiac mito-

chondrial function were determined.

Results Rats that received a high-fat-diet for 12 weeks had

increased body weight, visceral fat, plasma insulin levels, total

cholesterol, oxidative stress levels, depressed HRV, and cardiac

mitochondrial dysfunction. Garlic extract at both concentrations

significantly decreased the plasma insulin, total cholesterol,

homeostasis model assessment index, and oxidative stress lev-

els. Furthermore, garlic extract at both doses restored the HRV,

cardiac function, and cardiac mitochondrial function.

Conclusion We concluded that garlic extract at both

concentrations exerted cardioprotective effects against

cardiac dysfunction and mitochondrial dysfunction in

obese insulin-resistant rats.

Keywords Garlic extract � High-fat-diet � Insulin

resistance � Cardiac function �Mitochondrial function

Abbreviations

BCA Bicinchoninic acid

DCFDA Dichlorohydrofluorescein diacetate

ECG Electrocardiogram

EDP End diastolic pressure

EGTA Ethylene glycol bis (2-amino ethylether)-N,N,

N,N-tetraacetic acid

ESP End systolic pressure

HOMA Homeostasis model assessment

HPLC High-performance liquid chromatography

HR Heart rate

HRV Heart rate variability

JC-1 5,50,6,60-Tetrachloro-1,10,3,30-tetraethylbenzim-

idazolcarbocyanine iodide

MDA Malondialdehyde

DWm Mitochondrial membrane potential changes

ROS Reactive oxygen species

SV Stroke volume

TBA Thiobarbituric acid

Introduction

Long-term consumption of high-fat-diet is one of the

causes of obesity and has been shown to lead to insulin

L. Supakul � H. Pintana � N. Apaijai � S. Chattipakorn �K. Shinlapawittayatorn � N. Chattipakorn (&)

Cardiac Electrophysiology Research and Training Center,

Faculty of Medicine, Chiang Mai University, Chiang Mai 50200,

Thailand

e-mail: nchattip@gmail.com

L. Supakul � H. Pintana � N. Apaijai � K. Shinlapawittayatorn �N. Chattipakorn

Cardiac Electrophysiology Unit, Department of Physiology,

Faculty of Medicine, Chiang Mai University, Chiang Mai,

Thailand

S. Chattipakorn

Department of Oral Biology and Diagnostic Science, Faculty

of Dentistry, Chiang Mai University, Chiang Mai, Thailand

123

Eur J Nutr

DOI 10.1007/s00394-013-0595-6

resistance [1]. Insulin resistance is part of the metabolic

syndrome and can be characterized by hyperinsulinemia

with euglycemia [2, 3]. A previous study demonstrated that

insulin resistance can lead to oxidative stress as indicated

by increased reactive oxygen species (ROS) and malondi-

aldehyde (MDA) levels [4]. In addition, insulin resistance

has been shown to affect the heart by altering both the

mechanical function and cardiac autonomic balance. In the

past decades, heart rate variability (HRV) has been used to

indicate cardiac autonomic tone balance. Depressed HRV

has been shown to be associated with a poor prognosis in

post-myocardial infarction and heart failure patients [5].

Previous studies also demonstrated that obese insulin-

resistant rats induced by a high-fat-diet consumption

developed left ventricular dysfunction as well as depressed

HRV [2]. Moreover, cardiac mitochondrial dysfunction

was also observed in these obese insulin-resistant rats,

which could be responsible for decreased left ventricular

function.

Garlic (Allium sativum) has been used as a spice or

medicinal herb for many centuries [6]. It contains several

sulfur compounds including allylmethy, diallyl, dimethyl-

monotohexasulfide, diallydisulfide, allylmethyltrisulfide,

diallytrisulfide, vinyldithiins and ajoenes [7]. However, it

has been shown that the most biologically active com-

pounds contained within garlic are allicin [8] and several

sulfur-based compounds [9]. Garlic has been shown to

exert beneficial effects such as antibacterial [10] and

antifungal properties [11]. Interestingly, various studies

have reported that garlic also exhibits cardio protective

properties such as prevention of hyperlipidemia [12],

antithrombotic [13], and antiarrhythmic effects [14]. In

addition, garlic has been shown to increase the antioxidant

level in rats with cardio toxicity induced by doxorubicin

[15]. Despite these beneficial effects of garlic extract, its

cardioprotective effects in insulin resistance induced by

long-term high-fat-diet consumption are not well defined.

In the present study, we determined the effects of garlic

extract on the heart in obese insulin-resistant rats induced

by long-term high-fat-diet consumption. We hypothesized

that garlic extract administration could improve insulin

resistance and attenuate cardiac dysfunction and cardiac

mitochondrial dysfunction, which are impaired by long-

term high-fat-diet consumption.

Materials and methods

Animals and diet

All experiments were approved by the Institutional Animal

Care and Use Committees of the Faculty of Medicine,

Chiang Mai University, Chiang Mai, Thailand. Male

Wistar rats weighing 180–200 g were obtained from the

National Animal Center, Salaya Campus, Mahidol Uni-

versity, Thailand. Rats were kept at room temperature, 12-h

light/dark cycle and water ad libitum. Rats were randomly

divided into two groups to receive either a normal-diet or a

high-fat-diet (n = 24/group) for 12 weeks. In the normal-

diet group, rats were fed with standard laboratory chow

containing 19.77 % energy from fat. In high-fat-diet group,

rats were fed with diet containing 59.28 % energy from fat.

Then, rats in each group were divided into three subgroups

to receive one of the following treatments: vehicle (normal

saline), garlic extract 250, or 500 mg/kg/day (n = 8/

group). All rats received these treatments by intragastric

gavage for 28 days. The garlic extract solution was pre-

pared from commercially available garlic extract powder

capsules (Immunitop, Bangkok, Thailand) dissolved in

deionized water (2 ml/kg of body weight). Each capsule

contains 370 mg of garlic extract, which has 3,500 lg of

allicin, or approximately 1 % allicin in garlic extract per

capsule. Garlic extract solution was administrated once

daily via gavage feeding. Food intake was recorded every

day, and body weight was recorded weekly. Lead II elec-

trocardiogram (ECG) was recorded for HRV determina-

tion. Both ECG and blood samples were collected at

baseline, 4, 8, 12 weeks and at the end of treatment. After

28 days of treatment, rats were anesthetized and cardiac

function was determined by using the pressure–volume

catheter (Scisense, Ontario, Canada) [16]. At the end of

each study, the heart was removed and divided into two

parts for determining cardiac mitochondrial function and

cardiac MDA levels.

Plasma glucose, cholesterol, and insulin level

determination

Plasma glucose and total cholesterol levels were measured

by colorimetric assay using a commercial kit (Biotech,

Bankok, Thailand) [3]. Plasma insulin levels were mea-

sured by a sandwich ELISA kit (Linco Research, St.

Charles, MO) [3]. Insulin resistance was assessed by using

the Homeostasis Model Assessment (HOMA) which is a

mathematical model describing the degree of insulin

resistance. A higher HOMA index indicates a higher

degree of insulin resistance [3].

HRV analysis

Heart rate variability was used to determine cardiac sym-

pathovagal balance by using Power Lab (AD Instrument,

Sydney, Australia) and Chart 5.0 program [17]. Lead II

ECG was recorded for HRV analysis. In the present study,

frequency domain method was used to represent the HRV

and were determined using a MATLAB program [2, 18].

Eur J Nutr

123

High-frequency (HF) component generally represents car-

diac parasympathetic activity, whereas low-frequency (LF)

component usually represents cardiac sympathetic and

parasympathetic activity. LF/HF ratio was used to indicate

cardiac sympathovagal balance [19].

Cardiac and plasma MDA level determination

Cardiac and plasma MDA levels were determined by using

high-performance liquid chromatography (HPLC) base

assay [16]. Cardiac tissues were homogenized in phosphate

buffer pH 2.8. Plasma and cardiac tissues were mixed with

H3PO4 and thiobarbituric acid (TBA) to create TBA-

reactive substances. The plasma and cardiac TBA-reactive

substance concentrations were determined directly from a

standard curve and reported as MDA equivalent concen-

trations [20].

Cardiac function measurement

Rats were anesthetized via intramuscular injection using a

combination of zoletil (50 mg/kg, Virbac, Laboratories,

Carros, France) and xylazine (0.15 mg/kg, Laboratory

Carlier, SA, Barcelona, Spain). A ventral midline incision

at the neck was performed for tracheostomy. The rats were

ventilated with room air. The right carotid artery was

cannulated with a pressure–volume catheter (Scisense,

Ontario, Canada) for measuring left ventricular pressure

and volume for 20 min [2, 21, 22]. Cardiac function

parameters including heart rate, end systolic and end dia-

stolic pressure, maximum and minimum dP/dt, and stroke

volume were determined using the analytical software

program (Labscribe, Dover, NH) [2, 21, 22].

Cardiac mitochondrial isolation and mitochondrial

function determination

Cardiac mitochondrial isolation was performed as reported

previously [23]. The ventricles were removed and

homogenized in ice-cold buffer containing sucrose

(300 mmol/l), TES sodium salt (5 mM), and ethylene

glycol bis (2-amino ethylether)-N,N,N,N-tetraacetic acid

(EGTA) (0.2 mM), pH 7.2 (4 �C). The tissue was finely

minced and homogenized by using a homogenizer. After-

ward, the homogenate was centrifuged at 8009g for 5 min.

The supernatant was collected and centrifuged at

8,8009g for 5 min. The mitochondrial pellet was sus-

pended in ice-cold buffer and centrifuged once more at

8,8009g for 5 min. Protein concentration was determined

via the bicinchoninic acid (BCA) assay [23]. In this

study, cardiac mitochondrial function was determined

by measuring the mitochondrial ROS production, mito-

chondrial membrane potential changes, and mitochondrial

swelling. The morphology of cardiac mitochondria was

also determined using the transmission electron micro-

scope [2, 21, 22].

Determination of cardiac mitochondrial ROS

production

ROS was measured with the dye dichlorohydrofluorescein

diacetate (DCFDA) [24]. Isolated cardiac mitochondria

was incubated with two lM DCFDA at 25 �C for 20 min.

DCFDA fluorescence is excited at the wavelength of

485 nm, and the emission is detected at the wavelength of

530 nm. ROS concentration was determined using a fluo-

rescent microplate reader.

Determination of cardiac mitochondrial membrane

potential changes

Cardiac mitochondrial membrane potential changes (DWm)

were measured using the dye 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Isolated

cardiac mitochondria were stained with JC-1 (5 lM) at

37 �C for 30 min [25]. Mitochondrial membrane potential

was determined as fluorescence intensity and was measured

by a fluorescent microplate reader. JC-1 monomer (green)

fluorescence was excited at the wavelength of 485 nm and

the emission detected at the wavelength of 530 nm. JC-1

aggregate form (red) fluorescence was excited at the

wavelength of 485 nm and the emission detected at the

wavelength of 590 nm. The change in mitochondrial

membrane potential was calculated as the ratio of red to

green fluorescence intensity. The depolarization of cardiac

mitochondrial membrane potential was indicated by a

decreased red/green fluorescence intensity ratio [2, 23, 26].

Determination of cardiac mitochondrial swelling

Isolated cardiac mitochondria (0.4 mg/ml) was incubated

in 1.5 ml of respiration buffer (containing 100 mM KCl,

50 mM sucrose, 10 mM HEPES, 5 mM KH2PO4, pH 7.4 at

37 �C) [23]. Cardiac mitochondrial swelling was detected

using the spectrophotometer at a wavelength of 540 nm. A

decrease in the absorbance of the mitochondrial suspension

indicated mitochondrial swelling [23]. Transmission elec-

tron microscope was used to visualize the mitochondrial

morphology [3, 21].

Statistical analysis

All data were expressed as mean ± SE. One way ANOVA

followed by LSD post hoc test was used to determine the

difference between groups. P value \0.05 was considered

statistically significant.

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123

Results

Effects of high-fat-diet consumption on metabolic

parameters in rats

At the baseline, metabolic parameters were not signifi-

cantly different between the normal-diet group and the

high-fat-diet group (Table 1). After 12 weeks of high-fat-

diet consumption, body weight, plasma cholesterol, vis-

ceral fat, and plasma and cardiac MDA levels were sig-

nificantly increased, compared with the normal-diet-fed

rats. High-fat-diet-fed rats exhibited a significant increase

in plasma insulin level. Moreover, high-fat-diet-fed rats

also exhibited a significant increase in the HOMA index,

which is an indicator of insulin resistance (Table 1).

Effects of garlic extract on metabolic parameters

in obese insulin-resistant rats

In normal-diet rats treated with garlic extract at the dose of

250 and 500 mg/kg/day, the metabolic parameters in these

groups were not different from those in normal-diet rats

treated with vehicle (Table 2). In high-fat-diet-fed rats,

garlic extract at the dose of 250 and 500 mg/kg/day sig-

nificantly reduced plasma cholesterol, plasma insulin,

HOMA index, visceral fat, and plasma and cardiac MDA

levels, without altering plasma glucose, body weight, and

food intake (Table 2). The metabolic parameters in high-

fat-diet-fed rats treated with garlic extract of both doses

were not different.

Effects of garlic extract on HRV in obese insulin-

resistant rats

At the baseline, the LF/HF ratio was not different between

normal-diet- and high-fat-diet-fed rats. In high-fat-diet rats,

an increased in LF/HF ratio was observed at week eight

and reached the maximal level at week 12 (Fig. 1), indi-

cating depressed HRV. After the treatment of garlic extract

for 28 days at the dose of 250 and 500 mg/kg/day, the LF/

HF ratio was significantly decreased, compared with high-

fat-diet rats treated with vehicle (Fig. 2). There was no

Table 1 Metabolic parameters of normal-diet- and high-fat-diet-fed rats at baseline and at week 12

Metabolic parameters Baseline Week 12

ND HF ND HF

Body weight (g) 189 ± 4 190 ± 4 424 ± 9* 545 ± 18*,#

Food intake (g) 24 ± 0.31 25 ± 0.39 23 ± 1 23.92 ± 1

Plasma insulin (ng/ml) 1.59 ± 0.52 1.57 ± 0.42 1.93 ± 0.05 3.66 ± 0.95*,#

Plasma glucose (mg/dl) 137 ± 6 140 ± 13 147 ± 7 149 ± 5

HOMA index 14.16 ± 4.66 17.87 ± 3.16 16.68 ± 3.16 28.35 ± 2.16*,#

Plasma total cholesterol (mg/dl) 65 ± 13 63 ± 14 77 ± 2 105 ± 6*,#

Plasma MDA (lmol/ml) 2.08 ± 0.11 1.97 ± 0.16 2.17 ± 0.23 5.28 ± 1*,#

ND normal-diet, HF high-fat-diet

* P \ 0.05 versus Baseline, # P \ 0.05 versus ND week 12

Table 2 Effects of garlic extracts dose 250 and 500 mg/kg on metabolic parameters of normal- and high-fat-diet-fed rats

Metabolic parameters NDV NDG250 NDG500 HFV HFG250 HFG500

Body weight (g) 461 ± 11 467 ± 17 476 ± 16 567 ± 17* 571 ± 16* 574 ± 27*

Food intake (g) 22 ± 0.72 21 ± 0.79 21 ± 0.70 23 ± 0.59 23 ± 0.72 23 ± 0.35

Visceral fat (g) 27 ± 5 20 ± 2 22 ± 1 63 ± 4* 50 ± 3*,# 51 ± 3*,#

Plasma insulin (ng/ml) 1.97 ± 0.30 1.31 ± 0.63 1.56 ± 0.29 3.84 ± 0.39* 1.56 ± 0.1# 2.26 ± 0.41#

Plasma glucose (mg/dl) 155 ± 7 150 ± 13 142 ± 9 161 ± 12 149 ± 9 143 ± 14

HOMA index 16.19 ± 0.36 11.78 ± 3.44 13.79 ± 2.87 25.64 ± 1.52* 13.52 ± 1.29# 17.64 ± 3.13#

Plasma total cholesterol (mg/dl) 83 ± 7 76 ± 10 76 ± 4 161 ± 9* 84 ± 6# 79 ± 9#

Plasma MDA (lmol/ml) 1.96 ± 0.09 1.88 ± 0.02 1.87 ± 0.11 6.91 ± 0.04* 1.76 ± 0.04*,# 1.85 ± 0.06#

Cardiac MDA (lmol/mg protein) 5.94 ± 2.14 5.29 ± 0.82 5.54 ± 0.89 11.67 ± 2.53* 5.57 ± 1.16# 5.03 ± 0.62#

NDV normal-diet ? vehicle, NDG250 normal-diet ? garlic extract at dose 250 mg/kg, NDG500 normal-diet ? garlic extract at dose 500 mg/

kg, HFV high-fat-diet ? vehicle, HFG250 high-fat-diet ? garlic extract at dose 250 mg/kg, HFG500 high-fat-diet ? garlic extract at dose

500 mg/kg

* P \ 0.05 versus NDV, # P \ 0.05 versus HFV

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difference in the LF/HF ratio in high-fat-diet rats treated

with garlic extract of both doses. Furthermore, the LF/HF

ratio in the HF groups treated with garlic extract of both

doses was not different from the NDV group.

Effects of garlic extract on cardiac function in insulin-

resistant rats

In the normal-diet groups, heart rate (HR), end systolic

pressure (ESP), end diastolic pressure (EDP), -dP/dt, ?dP/

dt, and stroke volume (SV) were not different among

rats treated with vehicle, 250-mg/kg garlic extract, and

500-mg/kg garlic extract. In high-fat-diet rats, HR, EDP,

and -dP/dt were significantly increased, whereas the ESP,

?dP/dt, and SV were decreased, compared with rats on a

normal-diet. Interestingly, in high-fat-diet groups treated

with garlic extract at doses of 250 and 500 mg/kg/day, HR,

EDP, and -dP/dt were significantly decreased, whereas the

ESP, ?dP/dt, and SV were significantly increased, com-

pared with high-fat-diet rats (Table 3). There was no dif-

ference in the cardiac function parameters in the high-fat-

diet rats treated with garlic extract of both doses.

Effects of garlic extract on cardiac mitochondrial

function

Cardiac mitochondrial ROS production

In the normal-diet group treated with garlic extract at doses

of 250 and 500 mg/kg/day, the ROS level was not different

from that in the normal-diet group treated with vehicle

(Fig. 3). In the high-fat-diet group treated with vehicle,

ROS production was significantly increased compared with

the normal-diet group. In high-fat rats treated with both

garlic extract doses, the ROS level was significantly

decreased compared with the high-fat-diet group treated

with vehicle (Fig. 3). There was no difference in the ROS

production in the high-fat-diet rats treated with garlic

extract of both doses.

Cardiac mitochondrial membrane potential changes

(DWm)

In the normal-diet rats treated with garlic extract at doses

of 250 and 500 mg/kg/day, DWm were not different com-

pared to normal-diet rats treated with vehicle (Fig. 4). In

the high-fat-diet group treated with vehicle, DWm were

significantly decreased when compared with the normal-

diet group, indicating mitochondrial depolarization. In both

garlic-extract-treated groups, DWm were significantly

increased when compared with high-fat-diet group treated

with vehicle and was not different from the normal-diet

group (Fig. 4). There was no difference in the DWm in the

high-fat-diet rats treated with garlic extract of both doses.

Cardiac mitochondrial swelling

In the normal-diet group treated with garlic extract at doses

of 250 and 500 mg/kg/day, the absorbance was not dif-

ferent when compared with normal-diet-fed rats (Fig. 5). In

high-fat-diet group treated with vehicle, the absorbance

was significantly decreased when compared with normal-

diet group, indicating cardiac mitochondrial swelling.

However, in garlic-extract-treated groups, the absorbance

was restored back to a normal level (Fig. 5). There was no

difference in the absorbance in the high-fat-diet rats treated

Fig. 1 LF/HF ratio in normal-diet- and high-fat-diet-fed rats. LF/HF

ratio was increased at week eight and 12 of high-fat-diet consumption,

indicating impaired cardiac sympathovagal balance. LF/HF low-

frequency/high-frequency ratio, W4 week four of high-fat-diet con-

sumption, W8 week eight of high-fat-diet consumption, W12 week 12

of high-fat-diet consumption, ND normal-diet group, HF high-fat-diet

group. *P \ 0.05 versus baseline

Fig. 2 LF/HF ratio in normal-diet and high-fat-diet rats treated with

garlic extract at the doses of 250 and 500 mg/kg/day. Garlic extract at

both doses could reduce LF/HF ratio back to the normal level. LF/HF

low-frequency/high-frequency ratio, ND normal-diet group, HF

high-fat-diet group, NDV normal-diet ? vehicle, NDG250 normal-

diet ? garlic extract at the dose of 250 mg/kg, NDG500 normal-

diet ? garlic extract at the dose of 500 mg/kg, HFV high-

fat-diet ? vehicle, HFG250 high-fat-diet ? garlic extract at the dose

of 250 mg/kg, HFG500 high-fat-diet ? garlic extract at the dose of

500 mg/kg. *P \ 0.05 versus NDV, #P \ 0.05 versus HFV

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123

with garlic extract of both doses. Representative pictures of

cardiac mitochondria from an electron microscope are

shown in Fig. 6. Electron microscopic images revealed that

high-fat-diet caused unfolded cristae compared to normal

cardiac mitochondria, with marked swelling in the high-fat-

diet treated with vehicle group (Fig. 6d). Garlic extract at

both concentrations restored cardiac mitochondrial swell-

ing caused by high-fat-diet-induced insulin resistance

(Fig. 6e, f).

Discussion

The major findings of this study are as follows. In obese

insulin-resistant rats induced by long-term high-fat-diet

consumption, garlic extract could (1) improve metabolic

parameters and oxidative stress, (2) restore cardiac sym-

pathovagal tone balance, (3) prevent cardiac systolic and

diastolic dysfunction, and (4) restore cardiac mitochondrial

dysfunction. Previous studies demonstrated that long-term

high-fat-diet-fed rats for 12 weeks could cause insulin

resistance [1, 2, 26], by reducing the interaction between

insulin and insulin receptor substrate-1 (IRS-1), leading to

decreased insulin sensitivity and insulin resistance [27]. It

has been shown that insulin resistance can cause oxidative

stress by increasing ROS production leading to cardio-

vascular disease [28]. Since insulin resistance is strongly

associated with oxidative stress, reducing oxidative stress

under this condition has been shown to improve insulin

sensitivity as well as cardiac function [29, 30]. In the

present study, our findings that rats fed with high-fat-diet

for 12 weeks have developed insulin resistance as

Table 3 Effects of garlic extracts on cardiac function and hemodynamic parameters of normal-diet- and high-fat-diet-fed rats

Cardiac function NDV NDG250 NDG500 HFV HFG250 HFG500

HR(bpm) 324 ± 24 311.05 ± 40 333 ± 39 419 ± 3* 307 ± 27# 315 ± 19#

ESP(mmHg) 132 ± 8 145 ± 15 148 ± 12 92 ± 14* 134 ± 9# 141 ± 9#

EDP(mmHg) 16 ± 2 16 ± 0.02 15 ± 1.19 40 ± 3.12* 16 ± 0.28# 16 ± 0.23#

?dP/dt(mmHg/sec) 8,828 ± 43 9,830 ± 130 9,336 ± 351 5,410 ± 218* 9,894 ± 66# 9,612 ± 85#

-dP/dt(mmHg/sec) -5,960 ± 274 -5,030 ± 691 -5,240 ± 367 -3,809 ± 224* -5,074 ± 683# -5,334 ± 658#

SV(ll/g) 1.06 ± 0.04 1.03 ± 0.11 1.08 ± 0.07 0.74 ± 0.05* 0.94 ± 0.05# 0.99 ± 0.05#

NDV normal-diet ? vehicle, NDG250 normal-diet ? garlic extract at dose 250 mg/kg, NDG500 normal-diet ? garlic extract at dose 500 mg/

kg, HFV high-fat-diet ? vehicle, HFG250 high-fat-diet ? garlic extract at dose 250 mg/kg, HFG500 high-fat-diet ? garlic extract at dose

500 mg/kg

* P \ 0.05 versus NDV, # P \ 0.05 versus HFV

Fig. 3 Cardiac mitochondrial ROS production in normal-diet and

high-fat-diet rats treated with vehicle and garlic extract at the doses of

250 and 500 mg/kg. Garlic extract at both doses could reduce ROS

production in high-fat-diet-fed rats back to normal level. ROS reactive

oxygen species, ND normal-diet group, HF high-fat-diet group, NDV

normal-diet ? vehicle, NDG250 normal-diet ? garlic extract at the

dose of 250 mg/kg, NDG500 normal-diet ? garlic extract at the dose

of 500 mg/kg, HFV high-fat-diet ? vehicle, HFG250 high-fat-

diet ? Garlic extract at the dose of 250 mg/kg, HFG500 high-fat-

diet ? garlic extract at the dose of 500 mg/kg. *P \ 0.05 versus

NDV, #P \ 0.05 versus HFV

Fig. 4 Cardiac mitochondrial membrane potential changes in nor-

mal-diet and high-fat-diet rats treated with vehicle and garlic extract

at the doses of 250 and 500 mg/kg. Garlic extract at both doses

completely prevented cardiac mitochondria membrane depolarization.

ND normal-diet group, HF high-fat-diet group, NDV normal-

diet ? vehicle, NDG250 normal-diet ? garlic extract at the dose of

250 mg/kg, NDG500 normal-diet ? garlic extract at the dose of

500 mg/kg, HFV high-fat-diet ? vehicle, HFG250 high-fat-

diet ? garlic extract at the dose of 250 mg/kg, HFG500 high-fat-

diet ? garlic extract at the dose of 500 mg/kg. *P \ 0.05 versus

NDV, #P \ 0.05 versus HFV

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123

indicated by increased plasma insulin, cholesterol, HOMA

index, and MDA levels, without altering plasma glucose

level are consistent with previous reports [2, 3]. When

treated with garlic extract at 250 and 500 mg/kg/day, those

metabolic parameters were significantly improved, thus

indicating improved insulin sensitivity. These beneficial

effects of garlic extract are also consistent with previous

study that garlic extract could decrease insulin level, cho-

lesterol level, and oxidative stress in type 2 diabetes rats

[31]. Swanaton-Flatt et al. [32] reported that garlic extract

did not have hypoglycemic effect in streptozotocin-induced

diabetes rats. In the present study, both doses of garlic

extract did not have hypoglycemic effect in these obese

insulin-resistant rats either. Moreover, previous studies

have shown that garlic could decrease cholesterol con-

centration in obese rats [33, 34]. Padiya et al. [30] also

demonstrated that garlic homogenate could reduce insulin

level in type 2 diabetic rats after being treated for 8 weeks.

Furthermore, Banerjee et al. [35] reported that garlic has

powerful antioxidant ability to decrease the cardiac MDA

level in hearts with ischemic reperfusion injury.

In the present study, although insulin signaling was not

investigated, we demonstrated that garlic extract signifi-

cantly decreased oxidative stress in both plasma and car-

diac tissues, and this reduced oxidative stress was

associated with improved insulin resistance in our obese

insulin-resistant model. Furthermore, it has been previously

reported that garlic extract could affect the insulin signal-

ing pathway due to allicin. It is known that approximately

66 % of allicin in garlic can be decomposed to diallyl

disulfide (DADS) and hydrogen sulfide [36, 37]. Moreover,

hydrogen sulfide has been shown to improve glucose

uptake by increasing phosphorylation of the insulin

receptor, PI3K, AKT, and Glut 4 activation in insulin

signaling pathway [38, 39]. These beneficial effects of

garlic extract could be responsible for improved insulin

sensitivity in the present study.

In the present study, long-term high-fat-diet consump-

tion for 12 weeks caused not only insulin resistance, but

also cardiac sympathovagal tone imbalance (i.e., depressed

HRV) [1]. LF/HF ratio is one of several HRV parameters

that has been used to indicate cardiac sympathovagal tone

balance [5], in which increased LF/HF ratio indicates

depressed HRV [40]. Depressed HRV is the condition that

is normally observed in insulin resistance [2]. In the present

study, garlic extract at both concentrations restored HRV in

these obese insulin-resistant rats induced by long-term

high-fat-diet. A previous study has shown that insulin-

resistance-induced hyperinsulinemia could stimulate sym-

pathetic outflow, leading to HRV depression [41]. There-

fore, improved insulin sensitivity by garlic extract observed

in this study could be responsible for improved HRV. This

notion is supported by our findings that heart rate in high-

fat-fed rats were significantly higher than that in normal-

diet group and that garlic extract treatment could decrease

heart rate in these obese insulin-resistant rats.

In the present study, our obese insulin-resistant rats

developed cardiac contractile (both systolic and diastolic)

dysfunction, a similar finding reported previously [2].

Previous studies demonstrated that garlic oil can improve

cardiac function by increasing fraction shortening, ejection

fraction, and cardiac output in diabetes rats [42, 43]. A

growing number of studies have shown that garlic extract

exerts cardioprotective properties [44, 45] and has been

proposed to be due to its effect to increase cellular levels of

H2S that can prevent the progression of cardiac hypertro-

phy to heart failure [46]. In addition, garlic bioactives

primarily allicin and alliin have been shown to possess

cardioprotective properties [47–49]. In the present study,

our results demonstrated that garlic extract restored both

systolic and diastolic function that was impaired in these

obese insulin-resistant rats. In the present study, the role of

nitric oxide (NO) and hydrogen sulfide for the cardiopro-

tection of garlic extract was not investigated. Although

both NO and hydrogen sulfide have been shown as com-

ponents of active substances in garlic extract, the major

component that exerts many beneficial effects including

cardioprotection is ‘‘allicin’’ [48–51]. In the present study,

garlic extract with 1 % allicin was used and that this

concentration has been shown to provide cardioprotective

effects in previous reports [52, 53]. Furthermore, it has

been shown that allicin in garlic extract can be decomposed

to diallyl disulfide (DADS) [36] and that this DADS can be

changed to hydrogen sulfide [37], a product of allicin that

can also exert cardioprotection. Future studies will need to

Fig. 5 Cardiac mitochondrial swelling in normal-diet and high-fat-

diet rats treated with vehicle and garlic extract at the doses of 250 and

500 mg/kg. Garlic extract at both doses could prevent cardiac

mitochondrial swelling. ND normal-diet group, HF high-fat-diet

group, NDV normal-diet ? vehicle, NDG250 normal-diet ? garlic

extract at the dose of 250 mg/kg, NDG500 normal-diet ? garlic

extracts at the dose of 500 mg/kg, HFV high-fat-diet ? vehicle,

HFG250 high-fat-diet ? garlic extract at the dose of 250 mg/kg,

HFG500 high-fat-diet ? garlic extract at the dose of 500 mg/kg.

*P \ 0.05 versus NDV, #P \ 0.05 versus HFV

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explore possible role of NO and hydrogen sulfide on car-

dioprotection in this obese insulin-resistant model.

The heart is an organ that consumes much energy for

contraction and relaxation [54]. Cardiac mitochondria are

crucial organelles which generate energy for cardiomyo-

cytes [55]. In this study, we found that high-fat-diet con-

sumption for 12 weeks led to cardiac mitochondrial

dysfunction as indicated by increased mitochondrial ROS

Fig. 6 Representative pictures of cardiac ultrastructural morphology

by transmission electron microscopy (original magnification,

915,000). Normal cardiac mitochondria from the heart of the

normal-diet-fed rat is shown with apparent folded cristae (a). Cardiac

mitochondria from the heart of normal-diet-fed rat treated with garlic

250 mg/kg (b) and 500 mg/kg (c) also demonstrated apparent folded

cristae similar to that seen in (a). Cardiac mitochondrial swelling was

observed in the high-fat-diet-fed rat as indicated by unfolded cristae,

compared to that in the normal-diet group (d). Garlic extract at

250 mg/kg (e) and 500 mg/kg (f) preserved cardiac mitochondrial

morphology in the heart of obese insulin-resistant rats, as indicated by

clear folded cristae in cardiac mitochondria. ND normal-diet, G250

garlic extract at the dose of 250 mg/kg, G500 garlic extracts at the

dose of 500 mg/kg, HF high-fat-diet

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production, mitochondrial membrane potential depolariza-

tion, and mitochondrial swelling. We found that garlic

extract treatment restored cardiac mitochondrial function

by decreased mitochondrial ROS production and prevented

mitochondrial membrane depolarization and mitochondrial

swelling. The improved cardiac mitochondrial function by

garlic extract treatment in these obese insulin-resistant rats

could also be responsible for improved cardiac contractile

function found in this study.

In conclusion, long-term high-fat-diet consumption for

12 weeks could lead to insulin resistance, HRV depression,

cardiac contractile dysfunction, and cardiac mitochondrial

dysfunction. Treatment with garlic extract could attenuate

insulin resistance, HRV depression, cardiac dysfunc-

tion, and cardiac mitochondrial dysfunction in obese

insulin-resistant rats induced by long-term high-fat-diet

consumption.

Acknowledgments This work is supported by the Thailand

Research Fund Senior Scholar Grant RTA 5580006 (NC), BRG

5480003 (SC), and MRG5580125 (KS).

Conflict of interest The authors declare that they have no conflict

of interest.

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