Examination of the Relationship Between Intermittent Fasting and Irisin Levels in Rats Fed on a High-Fat Diet

INTRODUCTION: In this study, the relationship between intermittent fasting and irisin levels has been examined in rats fed on a high-fat diet.
18 wistar albino breed male rats were determined as the 1st control group (standard nutrition, 2.8% crude fat, 23.1% crude protein, 5% crude fiber, 7.1% crude ash and 12.8% moisture), the 2nd group was determined as the high-fat diet group (300 g/kg margarine was melted and added to standard pellet feed, prepared daily to be applied for 8 weeks daily), and the 3rd group was determined as the high-fat diet and intermittent fasting group (with a non-successive diet2 days a week, interrupted for 24 hours and all foodwasrestricted except water). As a result of the analyses performed, it was found that the level of the irisin in the group fed on a high-fat diet was low compared to the control group, and the level of the irisin in the high-fat diet + intermittent fasting group was high compared to the group fed on a high-fat diet. As a result, it was concluded that the use of intermittent fasting increases the level of irisin, and it can be considered among the methods to be used in the treatment of obesity to prevent its occurrence.

METHODS: Prior to the study, an approval was obtained fromDirectorate of Animal Experiments Local Ethics Committee, decision number 08, dated 25.06.2015. This study was conducted according to the Declaration of Helsinki, as revised in 2000. The study was conducted on 18 male wistar albino rats. All other conditions, except for the experimental diet, were provided within the standards of laboratory animal care. With 6 rats in each group,1st group was the control group (CG) and a standard feeding program was applied to this group (2.8% crude fat, 23.1% crude protein, 5% crude fiber, 7.1% crude ash, and 12.8% moisture) and the 2nd group was the high-fat diet (HFD) group (300 g/kg margarine was melted and added to the standard pellet feed, prepared daily and applied for 8 weeksdaily) and a high-fat diet with intermittent fasting (HFD+IF) was applied to the 3rd group (24 hours breakafter non-successive diet for 2 days a week, and all food was restricted except water). The study continued for a total of 8 weeks. At the end of the 8th week, a blood sample was taken from the heart of rats euthanized with Ketamine (50mg/kg) and the serum irisin level was examined via the ELISA method.
Taking of the blood samples: At the end of the experiment period, with general anesthesia application with ketamine (50mg/kg),the abdominal region of the rats (control and experimental groups) was excised in the form of an inverted letter V from the anal (pubis) area to the chest cavity, the abdominal cavity was opened, and the required amount of blood was taken by penetrating the heart with an injector. The blood taken was transferred to biochemistry tubes and centrifuged at 4000 RPM (RCF=1240xg)’’ for 15 minutes and serums were removed. Serums removed were placed in Eppendorf tubes and stored at -80°C until study.
Quantitative measurement of the irisinfrom the rat serum samples was performed using a commercial enzyme-linked immunosorbent analysis (ELISA) kit (Phoenix Pharmaceuticals Inc, Burlingame, California, USA). The determination range of the kit was 0.781 - 50 ng/ml, and intra-variation coefficients <0.469 and sensitivity and94% recovery values were measured using commercial enzyme-linked immunosorbent analysis (ELISA) kits (20).

Statistical Analysis
Descriptive statistics of the groups were given as mean and standard deviation. The Shapiro-Wilk test was used to determine whether the data were distributed normally or not. For the same parameter, presence of significant differences between the groups was evaluated with theKruskal-Wallis Test. In order to determine which group causes the difference, post hoc analysis (Tukey HSD) was performed and results which have a p value of 0.05 or smaller were considered significant.


RESULTS: Serum irisin values were lower in the high-fat diet group compared to the control group. Serum irisin values were higher in the high-fat diet + intermittent fasting group compared to the high-fat diet group (p<0.05) (Table 1).
DISCUSSION AND CONCLUSION: Irisin is an anti-obesity hormone that increases fat burning and prevents the formation of adiposity (21). Irisin, which was first isolated by Boström et al. in 2012, is a new myokine consisting of 112 amino acids (22). Irisin is also secreted in small amounts from other tissues, including liver or adipose tissue (23). Irisin is induced by exercise in rats and humans (24). The level of the irisinis increased with exercise and plays a significant role in increasing energy consumption by converting white adipose tissue into brown adipose tissue, allowing energy to be converted into heat (25). Irisin is a thermogenic agent and has a significant effect on reducing fat mass (23,26).
In their study, Mazur-Bialyet al. reported that plasma irisin levels were significantly reduced in sedentary rats fed on a high-fat diet (HFD) with or without colitis (27). In another study, Jimenez- Aranda et al. noted that there were high levels of irisin in thin rats, compared to obese rats (28).
Kanget al. found that in an 8-week high-fat diet study they conducted, the level of irisin was significantly reduced in the high-fat diet group compared to the control group after 8 weeks (29). In another study, Lu et al. showed that a high-fat diet increases body fat mass and reduces serum irisinlevels in wistar albino rats (30). It has been shown that the levels of irisin in the skeletal muscle and plasma of obese rats are reduced (22). Studies have shown that irisine in men is has a negative correlation with BMI, waist/hip ratio and fat ratio, and that the levels of the irisin were lower in obese and overweight men (31).
In another study, it was determined that FNDC5 decreases in rats fed on a high-fat diet, and the level of irisine in skeletal muscle also decreases significantly (32). These findings are supported by studies showing that irisin levels decrease during obesity in both rodents and humans (33,34). Stengel et al. reported that there is a positive relationship between body weight and circulating irisin in obese people (35). The results in the present studysupport the above-mentioned results and it has been shown that the level of irisin in the group fed on a high-fat diet was decreased significantly compared to the control group.
In the 7-week intermittent fasting study conducted by Karraset al, they found that the level of irisin increased after 7 weeks compared to the control group (36). In another study conducted by Alzoughoolet al., they found that fasting irisin values were higher compared to postprandial values during Ramadan (37). As a result of the eight-week study byCrujeriaset al., aiming to provide weight loss in 93 patients (38), the 10-week study by Huerta et al.,aiming to provideweight loss in 79 female patients (39),and the 6-week study of de la Iglesiaet al., aiming to provideweight loss, they all reported that in obese patients, with the decrease in the body weight,serum irisin levels were also decreased(40). In the study we carried out, it was observed that the level of the decreased irisin in the group fed on a high-fat diet was increased significantly in the group fed on a high-fat diet+ intermittent fasting.
In the literature search, it was observed that there are studies with different results on the levels of irisin in obese people. It is believed that the factor that causes inconsistency in irisin results is the biochemical analyzes (41). It has been stated that since irisin is a sensitive molecule,different results can be obtained in different kits, and the discrepancy in the study results may also be due to this (42).
In conclusion: In the present study carried out, it was seen that the 8-week intermittent fasting application with 2 days a week, which was applied to the group that was fed on a high-fat diet, increased the level of irisin, and it is contemplated that intermittent fasting can be employedas one of the methods of preventing and treating obesity,under the control of a physician, if necessary, by being supported with further studies on intermittent fasting.