Jurnal UMP Social Sciences and Technology Management
Vol. 3, Issue. 1,2015
The Effect of a Regular Aerobic Exercise Combined with
Consuming Garlic Extract on the Rest Levels of Plasma
Irisin and FNDC5 of the Heart Tissues and Muscles of Aged
Rats
Mahmood Jafari,
Department of Exercise Physiology,Faculty of Humaneties, Sari Branch, Islamic Azad University, Sari,Iran
Parvin Farzaneghi
Assistant Professor, Department of Exercise Physiology, Faculty of Humaneties, Sari Branch, Islamic Azad
University, Sari,Iran
[email protected]
Abstract
The aim of this study is to assess the effect of a regular aerobic exercise program combined
with consuming garlic on the rest levels of plasma irisin and FNDC5 of the heart tissue and
muscle of aged rats.
Methodology: In this study, 42 male aged rat (age=40 to 50 weeks, initial weight= 250-300gr)
were divided randomly into 6 groups with 7 rats in each group: 1) control group, 2) saline
group, 3) sham group 4) garlic group, 5) exercise group and 6) exercise-garlic group. The
exercise-garlic group received 1 ml garlic supplement for one kilogram of body weight through
oral gavage on daily basis. Swimming for 8 weeks (three days per week and 30 minutes in
every session) was considered as the exercise plan of this group. After the last round of
exercise session and following 10-12 hours of fasting the rats were anesthetized and their
plasma as well as heart tissue and muscle were removed immediately and kept in a freezer at
-70oc in order to measure plasma irisin and FNDC5 levels of heart and muscles .
Rat Irisin Elisa Kit was used to measure plasma irisin through ELISA. Data was analyzed
using independent t-test in order to determine the difference between groups. The
significance level was set at P≤0.05.
Findings: According to the findings, plasma irisin and FNDC5 levels were increased after 8
weeks compared with control group. But this increase was not significant (P>0.05 .)
Conclusion:According to results, swimming exercise for 8 weeks, consuming garlic and a
combined approach of exercise-garlic did not result in a significant increase in the plasma
irisin and FNDC5 levels of heart tissue and muscle in normal aged rats.
Keywords: swimming exercise, plasma irisin, aged rat, garlic extract
Introduction
As aging proceeds, elders encounter an ever-increasing risk of chronic diseases due to a
decreased level of physical activity and mobility originated from the nature of this period of
life. This leads to overweight and obesity problems associated with cardiovascular diseases,
diabetes, hypertension, chronic kidney diseases and other chronic diseases. Therefore, paying
attention to the life style of elders and their nutrition are of high importance. This becomes
more serious in developing countries due to lack of supporting programs or ineffective
programs affecting elders’ quality of life [5]. According to recent reports, many hormones
account for regulating energy cost and preventing overweigh. The hormones are released from
fat tissue and skeletal muscle. Some studies have concentrated on a number of proteins and
hormones that are produced in skeletal muscle and regulate energy cost and can be measured
both in skeletal muscle and blood [6]. Irisin is one of them. It is one of the newest discovered
hormones associated with energy homeostasis and consequent obesity [22]. Irisin has
significant and direct effects on fat tissue [21] and is considered as a potential indicator of
myocardial infraction [17]. Irisin is secreted into blood through the contraction of skeletal
286
The Effect of a Regular Aerobic Exercise Combined with ...
www.jsstm-ump.org
muscle and after releasing from type 4 phybronictoein protein membrane (including FNDC5
protein) [2]. Affecting white and brown fat tissue, this hormone increases energy consumption
and consequently decreases weight [6].Exercise triggers the production of irisin hormone and
FNDC5. This makes body cells to burn their excessive energy [18]. The hormones convert white
fat cells, accounted for saving energy, to brown fat cells. This is actually an approach by which
exercise can control body weight. It seems, however, irisin and FNDC5 help the prevention of
cell changes accounted for type 2 diabetes [16].According to studies, regular exercise positively
affects irisin and FNDC5 through increasing energy metabolism [7]. Moreover, it has been
accepted that as a mediator the hormones transfers the beneficial effects of exercise to the fat
tissues of human and rodents [9]. Sanchis-Gomar et al (2012) reported that irisin and FNDC5
are released from skeletal muscle. The extent of the release of both indicators can increase
energy cost and maintain blood glucose at a normal level [10]. In contrast, it has been shown
that irisin levels in blood flow, accounted for adjusting caloric cost, decreases in people who
perform exercise [11]. In addition, the attention of many researchers has been attracted towards
regular physical activity together with consuming herbs as factors improving quality of life and
body composition, preventing overweight and preventing and treating cardiovascular, kidney,
fat liver, overweight, metabolic disorders, like diabetes, and hypercholesterolemia diseases [12].
Garlic is a medicinal herb with an increased consumption rate aimed at treating and preventing
a large number of diseases. In some cases, it can be considered as a good substitution for drug
therapy [20]. It is a plant that strengthens body immune system with positive effects on some
types of cancer [19]. With its beneficial effects, it serves as an anti-spasm, anti-virus, antiseptic,
blood pressure reducer, anti-malaria, appetizer and bile stimulating herb [3]. Seo et al [2014]
showed that garlic and exercise did not affect the FNDC5 and plasma irisin levels of the skeletal
muscle of rats that are under high-fat diet. They accepted that adjusted FNDC5 and irisin levels
may not affect the inhibition of high-fat diet induced overweight and insulin resistance
through garlic supplement and exercise [15].
Based on the studies carried out on irisin and FNDC5 levels on the one hand and considering
the inattention of researchers to the effect of aerobic exercises on irisin and FNDC5 in elders on
the other hand, this study aims to answer this question: Does an 8-week aerobic exercise along
with consuming garlic have a significant effect on the plasma irisin and FNDC5 level of the
heart tissue and muscle of aged rats?
Methods
This is an experimental study because its population consists of randomly selected Wistar male
rats and many variables of the trials were under control in the laboratory. In this study, 42 aged
male rats (age= 40 to 50 weeks; initial weight= 250-300 gr) were divided into 6 groups: 1)
control group, 2) saline group, 3) sham group, 4) aerobic exercise group, 5) garlic group and 6)
garlic-exercise group.During the study phases, the studied rats were kept in transparent
polycarbonate cages (size=30*15*15) made by Razi Rad Co. in the following condition:
oc
oc,
light/dark cycle ratio (LDR): 12h: 12h, temperature=22 ±2 humidity= 50%±5% and ventilation
condition= acceptable. The food of trials was produced by Karaj Dam Behparvar Food Co. and
was put in every cage based on the weight of rats that were measured once three days with
standard weighing machine and considering the natural share of 10 gr per 100 gr of body
weight. In all stages, water was supplied freely to rats within animal-specialized bottles with a
volume of 500 ml.First of all, old garlic was supplied from market and was cleaned and ground.
It was kept for 3 hours in room temperature and humidity and extraction process was
performed through maceration method. To start extraction process, a total of 50 gr ground
garlic was poured in a 1 liter balloon and methanol was added up with a ratio of 1:3. The
solution, then, was filtered by Buchner funnel and methanol was added up to the remained
slag. The slag was filtered again after 24 hours and was added to the first extract. The obtained
oc
solution, then, was distilled in a vacuum distillation device at a temperature of 50 and at a
speed of 70 rpm until the solution volume was decreased to 1/5 of its initial volume. At this
point, the extract tank was separated from the device. When the solution was cooled it was
287
Jurnal UMP Social Sciences and Technology Management
Vol. 3, Issue. 1,2015
decanted for three times with 50 ml chloroform. The remained volume was poured in a Petri
oc
dish with a given weight and was ovened within device at 50 . When the extract was dried, it
was weighted and every ¼ gram of the extract powder was mixed with 56 ml distilled water.
The supplement group and supplement-exercise group were received 1 ml garlic extract for
every kilogram of their body weight on daily basis for 8 weeks through oral gavage. Saline
group, however, was received the same volume of saline supplement in the same manner [13].
Before the commencement of the main protocol, the trials of exercise group were trained for
one week (five days per week and each time for five minutes) in order to learn swimming. The
main exercise plan was as follows: swimming for 8 weeks (three days per week and 30 minutes
oc
oc
for every session [13]) in water (temp=32 ±2 ). A five minutes interval was considered before
and after the main exercise in order to let the trials warm and cool. Blood sample collection
and biopsy operations were conducted 48 hours after the last exercise session and after 10-12
hours of fasting. The rats were anesthetized by intra peritoneal injection of Ketamin (30-50
mg/kg) and Xylazine (3-5 mg/kg). Then, their blood, heart and soleus muscles were
oc
immediately removed and placed in a freezer at -70 for measuring irisin and FNDC5 levels
purposes. To avoid the influence of circadian heart rhythm rate, sampling operation was
started at 8:00 a.m. and was completed at 11:30 a.m.
Measurement of Variables
A total of 0.5 gr of the collected tissue (heart and muscle) was placed in liquid nitrogen. The
tissue, then, was homogenized in 1 ml phosphate buffered saline 1x with 1mM PMSF in a
oc
oc
homogenizer device at 0 . The obtained solution was centrifuged for 10 minutes at 4 (speed=
4000 rpm). In the next step, the outer layer of solution was filtered and used in ELISA test. The
concentration of plasma irisin was measured through ELISA method using Rat irisin ELISA Kit,
Sunlong Biotch, China with a sensitivity of 1 pg/ml. However, the concentration of FNDC5 of
heart and soleus muscle was measured using the ElISA Kit of Biotech Rat Fibronectin Type III
Domain Containing Protein 5 (FNDC5) ELISA Kit, China with a sensitivity of 1.5
pg/ml.Descriptive statistics was used to determine and classify the index of dispersion. Levene
test and ANOVA were used to assess the equality of variables variance and the change between
groups, respectively. Tukey test was used to determine differences between groups. In this
assessment, sig. level of P≤0.05 was considered as the measure for rejecting the null hypothesis.
All statistical calculations were conducted in SPSS 16 and all graphs were drawn in Excel.
Findings
According to ANOVA analysis, there was a significant difference in plasma irisin between the
studied groups after 8 weeks so that Tukey test results showed a significant increase of irisin in
exercise group (6.73%), garlic group (5.97%) and exercise-garlic group (6.39%) compared with
control group (see Fig. 1). However, no significant difference was seen in the serum levels of
irisin between exercise-garlic and control groups (P>0.05).
Fig. 1: mean level of plasma irisin in the studied groups. * indicates a significant difference
compared with control, saline and sham groups
288
The Effect of a Regular Aerobic Exercise Combined with ...
www.jsstm-ump.org
According to ANOVA analysis, there was no significant difference in the FNDC5 level of heart
tissue between the studied groups after 8 weeks. Although there was an increase in exercise
group (11.46%), garlic group (7.90%) and exercise-garlic group (7.11%) compared with control
group (see Fig.1), this difference was not statistically significant (P>0.05) (see Fig. 2)
Fig. 2: mean level of FNDC5 protein in the heart tissues of the studied groups
According to ANOVA analysis, there was no significant difference in the FNDC5 level of muscle
tissue between the studied groups after 8 weeks. Although there was an increase in exercise
group (19.86%), garlic group (17.21%) and exercise-garlic group (15.89%) compared with control
group, this difference was not statistically significant (P>0.05) (see Fig. 3)
Fig. 3: mean level of FNDC5 in the muscle tissue of the studied groups
289
Jurnal UMP Social Sciences and Technology Management
Vol. 3, Issue. 1,2015
Discussion
As aging proceeds, elders encounter an ever-increasing risk of chronic diseases due to a
decreased level of physical activity and mobility originated from the nature of this period of
life.This leads to overweight and obesity problems associated with cardiovascular diseases,
diabetes, hypertension, chronic kidney diseases and other chronic diseases. Therefore, it is very
important to pay attention to one of the most important issues of this period of life, especially
in developing countries [5]. The attention of researchers has been attracted to performing
regular physical activities together with consuming herbs as factors improving quality of life
and body composition, preventing overweight and preventing and treating cardiovascular,
kidney, fat liver, overweight, metabolic disorders like diabetes and hypercholesterolemia [12].
The researchers stated that exercise is an important factor affecting the secretion of this
hormone and the other effects of this hormone on energy metabolism. However, the type of
exercise and the minimum duration of exercise required for physical health are not clear yet
[8].The type of this study and the specifications of the trials differentiate it from other studies.
Roka et al (2013) showed that short-term resistance training stimulates the secretion of FNDC5
in hypodermic and visceral adipose tissue. They observed that white adipose tissue (WAT)
decreases the secretion of FNDC5 in non-fed animals. The white fat tissue of fat animals creates
a kind of resistance through the excessive secretion of this hormone as 72% of total secretion of
FNDC5 and irisin is attributed to muscle-secretion [14].Raeisi et al (2013) found that plasma
irisin level increases significantly following a resistance training session. However, there was a
significant increase in the relative expression of FNDC5 and UCP1 genes following exercise.
They concluded that by secreting myokines like irisin, resistance training may improve body
composition through increased conversion of white fats to brown fats [4].It is likely, therefore,
that the resistance training of this study could not apply the required pressure on muscles in
order to stimulate the secretion of myokines like irisin and FNDC5. A brief look at the findings
of this study reveals that consuming garlic has not a considerable effect on the studied
indicators in mice.
Conclusion
The results of this study showed that following an 8-week aerobic swimming exercise, there is
an increase in the levels of plasma irisin and FNDC5 of rats’ heart tissue and muscle but this
increase is not significant. Therefore, it is suggested to future studies to more concentrate on
the effect of different types of exercise on plasma irisin variations.
References
1- Khodadadi. Hashem, Rajabi. Hamid, Attarzde Hoseini. Seyyed Reza, Abbasian. Sadegh: “effects of acute periodical
exercise and Pilates on the serum level of irisin and insulin resistance of obese women”; Iranian journal of endocrine
glands and metabolism, 2014- period 16, No. 3, pgs. 190-196
2- Shirzad. Hedayatollah, Taji. Fateme, Pilevarian. Aliasgar, Hoseini. Seyyed Masih, Rafieian Koupaei. Mahmoo:
“preventive and remedial effects of fresh garlic essence and aerial parts on the growth of fibrosarcoma cells of mouse,
2013. Scientific-Research Journal of Shahid Sadougi University of Medical Sciences-Yazd, Period 20, No. 2, pgs 133-141
3- Rambod. Masume, Rafiei. Forough, Khabbaz Shirazi. Mohammad, Ghodsbin. Fariba, Heidari. Seyyed Taghi:
“comparing the life quality of elders with chronic kidney disorders with young and middle-aged people”, year 6. No. 19,
pgs 52-58
4- Raeisi. Jalil, Rajabi. Hamid, Ghaedi. Kamran, Marandi. Seyyed Mohammad, Dehkhoda. Mohammadreza: “effect of
acute resistance training on the level of plasma irisin and expression of FNDC5 muscle gene and UCP1 tissue in male
rats”, 2013, Journal of Isfahan University of Medical Sciences. 31 (256), 1657-1666
5- Jacques R, Poortmans and michel ouchinskg. Glomerular filtation Rate and Albumin Excretion After Maximal
Exercise in Aging Sedentary and Active men. J Geron. 2006; 11 (61):1181-1185.
6- Finkelstein J, Joshi A, Hise MK. Association of physical activity and renal function in subjects with and without
metabolic syndrome: a review of the Third National Health and Nutrition Examination Survey (NHANES III). Am J
Kidney Dis 2006; 48: 372–382.
7- Johansson K, Neovius M, Hemmingsson E. Effects of anti-obesity drugs, diet, and exercise on weight-loss
maintenance after a very-low-calorie diet or low-calorie diet: a systematic review and meta-analysis of randomized
controlled trials. Am J Clin Nutr 2014;99:14–23.
8- Ormsbee MJ, Thyfault JP, Johnson EA, Kraus RM, Choi MD, Hickner RC. Fat metabolism and acute resistance
exercise in trained men. J Appl Physiol (1985 ) 2007; 102(5): 1767-72.
390
The Effect of a Regular Aerobic Exercise Combined with…
www.jsstm-ump.org
9- Moreno-Navarrete J. M, F. Ortega, M. Serrano, “Irisin is expressed and produced by human muscle and adipose
tissue in association with obesity and insulin resistance,” Journal of Clinical Endocrinology & Metabolism, vol. 98, no.
4, pp. E769– E778, 2013.
10- Powolny AA, Singh S. Multitargeted prevention and therapy of cancer by diallyl trisulfide and related Allium
vegetable-derived organosulfur compounds. Cancer letters 2008; 269(2): 305-314.
11- Polyzos SA, Kountouras J, Shields K, Mantzoros CS. Irisin: a renaissance in metabolism?. Metabolism 2013;62:1037–
1044.
12- Cunha A. Basic research: Irisin--behind the benefits of exercise. Nat Rev Endocrinol 2012;8:195.
13- Ashraf R, Aamir K, Shaikh AR, Ahmed T, Effects of garlic on dyslipidemia in patients with type 2 diabetes mellitus. J
Ayub Med Coll Abbottabad 17 (2005) 60–64.
14- Roca-Rivada, C. Castelao, L. L. Senin., “FNDC5/irisin is not only a myokine but also an adipokine,” PLoS One, vol.
8, Article ID e60563, 2013.
15- Seo DY, Kwak HB, Lee SR, Cho YS, Song IS, Kim N, Bang HS, Rhee BD, Ko KS, Park BJ, Han J. Effects of aged garlic
extract and endurance exercise on skeletal muscle FNDC-5 and circulating irisin in high-fat-diet rat models. Nutr Res
Pract. 2014;8(2):177-82.
16- Moinuddin, Leehey DJ. A coparison of aerobic exercise and resistance training in patints with and without chronic
kidney disease. Adv chronic kidney Dsi. Jan ; 2008, 15 (1): 8317- Kuloglu T,Aydin S, Eren MN, Yilmaz M, Sahin L, Kalayci M, Sarman E, Kaya N, Yilmaz OF, Turk A, Aydin Y, Yalcin
MH,Uras N, Gurel A, llhan S,Gul E, Aydin S . Peptides.2014 MAY;55:85-91.doi:10.1016/j.peptides.2014.02.008. Epub 2014
feb 24
18- Moinuddin, Leehey DJ. A coparison of aerobic exercise and resistance training in patints with and without chronic
kidney disease. Adv chronic kidney Dsi. Jan; 2008, 15 (1): 83-96.
19- MacIntosh B, Gardiner P, McComas A. Skeletal muscle: form and function. Trans. Gharakhanlo R, Azad A, Gorzi A.
Tehran, Iran: Samt Publication; 2011. [In Persian].
20- Marta G. Novelle, Cristina Contreras, Amparo Romero-Picó, Miguel López and Carlos Diéguez. Irisin, Two Years
Later (Review Article). International Journal of Endocrinology. 2013; 10(8): 1-8.
21- Boström P, Wu J, Jedrychowski MP, Korde A, Ye L, Lo JC, Rasbach KA, Boström EA, Choi JH, Long JZ, Kajimura S,
Zingaretti MC, Vind BF, Tu H, Cinti S, Højlund K, Gygi SP, Spiegelman BM. A PGC1-alpha-dependent myokine that
drives brown-fat-like development of white fat and thermogenesis. Nature 2012;481:463–468.
22- Pedersen BK. A muscular twist on the fate of fat. N Engl J Med 2012; 366(16): 1544-5.
391