Abstract
Background:
The UCP2 gene variation has been associated with the increased risk for obesity and lower response to an energy-restricted diet.
Objectives:
This study aimed to compare the effect of intermittent fasting diet (IFD) and low-calorie diet with exercise (LCD-ex) based on UCP2 gene variations.
Methods:
Subjects were adult men and women (n = 125) with body mass index between 25–40 kg/m2. During IFD, subjects were abstained from eating and drinking for 13 hours. During LCD-ex, subjects received dietary counseling for low-calorie diet and group exercise program. Anthropometric changes were compared after 4 weeks. The -866 G/A (rs659366) UCP2 gene variation was analyzed and subjects were separated into 2 groups: GG and AA+GA.
Results:
In GG genotype, IFD was associated with a higher weight reduction compared to LCD-ex (–1.7±0.2 vs. –0.7±0.3 kg, p = 0.016). In AA+GA genotypes, there were no differences in weight loss between IFD and LCD-ex (–1.7±0.3 vs. –1.7±0.3 kg, p = 0.967). No differences in energy intake between genotypes (all p > 0.05).
Conclusions:
This study shows that GG genotype of -866 G/A UCP2 was associated with a better response towards IFD compared to LCD-ex.
Introduction
Obesity has been associated with the increased risk for non-communicable diseases such as type 2 diabetes mellitus, hypertension and cardiovascular diseases [1], thus an effective weight loss treatment is highly warranted [2]. Lifestyle interventions including alteration in physical activity and dietary interventions have been developed and applied worldwide. However, it was very unfortunate that not all individual can rely on the current interventions for an effective weight loss regime [3]. It has been suggested that genetic variations play an important role in an individual’s response towards a weight loss program [4].
Uncoupling protein (UCP) is a protein that has an important role in mitochondrial energy metabolism [5]. UCP2 is one of the UCP subtypes which present in the majority of human tissues and has been linked with efficiency of energy metabolism [6]. It has been reported that UCP2 gene polymorphism was associated with the increased risk of obesity [7–9]. Additionally, studies showed that UCP2 is associated with the differential response to an energy restriction diet [10, 11]. We selected the gene variation at promoter region –866 (rs659366) because it was previously reported that G/A variation in this region was not only associated with UCP2 gene expression but also the metabolic rate in humans [12, 13].
In this study, we are interested to introduce an intermittent fasting diet as an alternative solution for those who own UCP2 gene variation that had lower response towards a conventional low calorie diet. Intermittent fasting diet is a time-based energy restriction regime in which an individual abstains from eating for a restricted period of time [14]. Several studies have shown that intermittent fasting diet had beneficial effects on weight loss for obese individuals [15–17]. One of the examples of the intermittent fasting is the restrictive diet during Ramadan which usually conducted by Moslems all around the world.
The aim of this study was to compare individual responses towards intermittent fasting diet (IFD) versus conventional low-calorie diet with exercise (LCD-ex) on body weight and composition based on -866 G/A (rs659366) UCP2 gene variation. Intermittent fasting diet was observed in individuals with overweight and obesity who undertook a Ramadan fasting. We hypothesize that individuals with overweight/obesity responded differently on IFD and LCD-ex based on -866 G/A UCP2 gene variation. To our knowledge, there were no previous reports in the investigating response of intermittent fasting diet in UCP2 gene polymorphism.
Materials and methods
This is a secondary data analysis of the role of UCP2 genetic variation on individual’s response to two dietary alteration regimes in two sub-studies: an intermittent fasting diet study (IFD) and a low calorie diet with exercise study (LCD-ex). These sub-studies were conducted in Yogyakarta, Indonesia in different months but in the same years (2015 and 2017). The intermittent fasting study was conducted in June-July during Ramadan fasting period while low calorie diet with exercise study was conducted in September-October. Subjects were overweight/obese men and women adults. The inclusion and exclusion criteria of subjects were similar for those 2 sub-studies. The inclusion criteria were age between 21–56 years old, body mass index (BMI) between 25 and 40 kg/m2, and not diagnosed with chronic diseases by medical doctors. Subjects who were pregnant, breastfeeding, consuming any long-term medication, current and past smokers and had problems with walking or conducting physical activity in the last 6 months were excluded from this study. Ethical clearance for both studies (intermittent fasting diet: KE/0377/03/2017; low-calorie diet with exercise KE/0560/05/2017) were obtained from from the Medical and Health Research Ethics Committee, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Indonesia.
Sub-study I: The intermittent fasting diet (IFD)
The sub-study I (IFD) was an observational study with cohort design in which subjects were followed during the Ramadan fasting period. Subjects were selected using a purposive method. We followed 177 adults living in the city of Yogyakarta during Ramadan fasting period. Of those, 127 subjects were measured at the end of the fasting period but only 67 of them had BMI between 25–40 kg/m2. Data on dietary intake and anthropometric measures were collected before and after 4 weeks of the fasting period.
All subjects in this study were Moslems. During the Ramadan fasting period, all subjects abstained from eating and drinking from dusk until dawn. In Indonesia, this fasting period usually started around 4.30 am and lasted until 6.00 pm (approximately between 13 and 14 hours-per-day). Those eating and drinking restrictions had an impact on the reduction of meal frequency from 3 times a day to 2 times a day. During the fasting period, subjects can still conduct their usual physical activity. During IFD, subjects did not received any dietary counseling.
Sub-study II: The low calorie diet with exercise (LCD-ex)
The sub-study II (LCD-ex) was an intervention study using a lifestyle-based weight loss program which consists of diet and exercise. Fliers and announcements were released around the university and we recruited those who were interested in losing weight. Informed consent was given by those who agreed to participate in this study. Subjects received dietary counselling for weight loss using a low calorie - balanced nutrients diet regime (1200–1500 kcal/day). The counselling sessions were conducted privately between subjects and nutritionists who hired for this study. The dietary counselling was given 2 times: at the first day of the intervention and 2 weeks at intervention to check whether subjects complied with the diet. Subjects in this sub-study also followed an exercise program. The exercise program was a combination of aerobic exercise and resistance exercise and conducted 2 times a week with duration of 60 minutes each session.
Every 2 weeks, subjects were measured for their anthropometric profile and the main purpose of this sub-study was to evaluate the effect of LCD-ex until 8 weeks. However, for the purpose of this study, we compared the data at the baseline and after 4 weeks of the intervention to match the amount of weeks from those who participated in the IFD (sub-study I). A total of 93 adults initially involved in the program but only 58 subjects with BMI between 25–40 kg/m2 and measured at week 4.
Anthropometric measures
Body weight was measured using a digital body mass scale (0.01 kg) (Omron, Japan) and height was measured using a wall-mounted height rod (0.1 cm). The overweight and obesity statuses were defined by BMI, which was calculated by dividing body weight (in kg) to the square of height (in m). Percent body fat was measured using a bio-electrical impedance analysis (Omron, Japan). Fat-free mass was calculated by reducing actual body weight (in kg) with body fat (in kg). Those anthropometric measurements were done by trained enumerators using calibrated instruments. Data of anthropometric measures were recorded twice: at the baseline and after 4 weeks.
Dietary intake and physical activity analysis
Data of dietary intake including total energy intake (in kcal) and macronutrients (protein, fat, and carbohydrate) were extracted from food consumption data. Information on food consumption was recorded using a semi-quantitative food frequency questionnaire which represented subject’s dietary habit. This data collection was conducted via a face-to-face interview done by trained enumerators. Energy intake was calculated based on total energy (in kcal) that was provided by consumed food items. Intake of macronutrients, such as protein, fat, and carbohydrate, were also analyzed. Data of dietary intake were recorded twice: at the baseline and after 4 weeks (which represent dietary intake during the intervention / cohort period). Physical activity was measured using global physical activity questionnaire (GPAQ) which was previously translated, validated and used in Indonesia [18].
Genotype analysis
DNA sample was collected from subjects’ blood specimen. Blood was drawn and directly located into EDTA tube. After centrifugation of the blood at 1500 rpm for 10 minutes, the buffy coat was separated from the whole blood. The DNA sample was isolated from buffy coat using a commercial DNA extraction kit (Favorgen, Taiwan). UCP2 -866 G/A (rs659366) genotyping was done using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with forward primer: 5′-CAC GCT GCT TCT GCC AGG AC-3′ and reverse primer: 5′-AGG CGT CAG GAG ATG GAC CG-3′ [19]. PCR conditions are 8 minutes of denaturation in 95°C followed by 35 cycles of 95°C for 1 minute (denaturation), 55°C for 1 minute (annealing), 68°C for 1 minute (extension) and 72°C for 7 minutes (final extension). The PCR product then was digested using BST UI enzyme digestion. Restriction fragments were resolved on a 3% agarose gel.
Statistical analysis
Statistical analysis was done using JASP software (The University of Amsterdam). Prior to analysis, all datasets were checked based on their normality of distribution using the Shapiro Wilk test. Subjects were separated based on their UCP2 genotypes group GG vs AA+GA. This separation was because previous study showed that GG genotype had different UCP2 expression compared to those with A allele carriers (AA+GA) [20]. Baseline anthropometric data were compared between IFD and LCDex gene-specific groups (GG vs AA+GA) using an independent t-test for normally distributed data and Mann-Whitney test for not normally distributed data. Changes in dietary intake and anthropometric measures before and after 4 weeks were calculated by simple reduction except for changes in body weight. Changes in body weight were analyzed using its absolute changes value (in kg) and relative changes based on their initial body weight (%). This was done to correct the influence of initial body weight on weight changes. The impact of each dietary regime on changes in body weight and body composition between UCP2 genotypes were analyzed using an independent t-test or Mann-Whitney test.
Results
This is a secondary analysis of 2 different sub-studies. The first study (IFD) investigated the effect of UCP2 gene variation on weight loss during the Ramadan fasting period (4 weeks) and the second study (LCD-ex) was an intermediate analysis of anthropometric measures after 4 weeks of the low-calorie diet with exercise. Subjects in this study were selected from those with BMI between 25 and 40 kg/m2. The purpose of this selection was to eliminate the effect of variation in their initial body weight by excluding those with BMI higher than 40 kg/m2. Characteristics of subjects are presented in Table 1. In this study, we found that there were no differences in age, height, baseline anthropometric, physical activity and baseline dietary intake between UCP2 genotype groups.
Baseline characteristics of subjects with different variation of UCP2 gene
Baseline characteristics of subjects with different variation of UCP2 gene
†Mann-Whitney test; 1percent weight changes compared with in itial weight changes; IFD: intermittent fasting diet; LCD-ex: low calorie diet with exercise; data are presented as mean±standard error of mean.
Differences in dietary intake during the intermittent fasting (IFD) and low-calorie diet with exercise (LCD-ex) are presented in Table 2. In this study, we found that there were no differences in energy intake between IFD and LCD-ex in GG genotype (p = 0.637) and in AA+GA genotypes (p = 0.224). We also reported no significant differences in protein intake, carbohydrate intake and physical activity between IFD and LCD-ex in GG genotype and AA+GA genotypes (all p > 0.05). However, we found that dietary fat was higher among those who took IFD than those who took LCD-ex in both genotype groups (all p < 0.05).
Comparison on dietary intake between UCP2 genotypes during intermittent fasting (IF) and low calorie diet with exercise (LCD-ex) at week 4
IFD: intermittent fasting diet; LCDex: low calorie diet with exercise; data are presented as mean±standard deviation. ‡ Independent t-test; †Mann-Whitney test; pGG: comparison between IFD and LCDex in subjects with GG genotype; pAA +GA: comparison between IFD and LCDex in subjects with AA+GA genotype; pIFD: comparison between AA+GA and GG genotypes during IFD.
The interaction between UCP2 gene variations and the dietary regime was analyzed by grouping the subjects based on their UCP2 gene variation (Table 3). In subjects with AA or GA genotypes, there were no differences in body weight and body composition changes after LCD-ex or IFD (p = 0.967). For subjects with GG genotype, weight loss response towards IFD was higher than towards LCD-ex (p = 0.016). The higher response towards IFD than LCD-ex in GG genotype of UCP2 was confirmed by the difference in percent body weight compared to initial body weight (p = 0.020). We reported no interaction between UCP2 and the dietary regimes on percent body fat or fat-free mass index (all p > 0.05).
Differences in anthropometric measures between UCP2 genotypes during intermittent fasting (IFD) and low calorie diet with exercise (LCD-ex)
1percent weight changes compared with initial weight changes; IFD: intermittent fasting diet; LCDex: low calorie diet with exercise; data are presented as mean±standard deviation. ‡ Independent t-test; †Mann-Whitney test; pGG: comparison between IFD and LCDex in subjects with GG genotype; pAA +GA: comparison between IFD and LCDex in subjects with AA+GA genotype; pIFD: comparison between AA+GA and GG genotypes during IFD.
The additional analysis was to measure the effect of confounding factors towards the weight changes. In all subjects, age (r = –0.099, p = 0.274), initial body weight, (r = –0.117, p = 0.193), initial BMI (r = 0.124, p = 0.169) and initial energy intake (r = –0.052, p = 0.568) were not correlated with weight loss. There were no significant differences in percent weight loss between men and women in this study (p = 0.197). We found no correlation between physical activity during IFD and LCD-ex on weight changes in all groups (p = 0.850) and in genotype specific groups (AA+GA p = 0.966; GG p = 0.897). The influence of potential confounding factors, i.e. age, sex, initial body weight, initial BMI and energy intake were not seen in genotype-specific groups (all p > 0.050) (data are not shown).
This study aimed to compare the differential response towards two dietary regimes in overweight/obese individuals based on UCP2 gene variations. Although subjects in IFD and LCD-ex had similar energy intake and physical activity after 4 weeks, we showed that they reduced body weight differently according to UCP2 gene variation. Subjects with GG genotype of -866 G/A UCP2 had lower response towards LCD-ex compared to those with AA+GA genotypes. However, subjects with GG genotype who undertook IFD had a significantly higher weight loss compared to those who undertook LCD-ex.
The importance of UCP2 gene on weight loss in an hypo-caloric based intervention has been recently highlighted by Cortes-Oliveira et al. [11]. In the study, they showed that UCP2 gene expression was positively correlated with weight loss during the intervention. Several studies suggested that genetic variation of UCP2 in other locations were associated with differential response towards diet. To our knowledge, currently, there is no study reported the impact of -866 G/A UCP2 gene polymorphism on individual responses to an intermittent fasting diet. Cha et al. [21] showed an association between Ala55Val gene variation in UCP2 and response to an energy-restricted diet in a 1-month intervention study. A study in Japanese adults showed that I/D gene polymorphism of UCP2 was associated with changes in BMI after a 3-month intervention program. It was suggested that this effect was due to the impact of UCP2 gene variation on differences in energy expenditure [21].
Because the GG genotype was associated with lower weight reduction compared to AA+GA genotypes during low-calorie diet regime, we assumed that the mechanism was similar to those reported by Mutombo et al. [10]. It was previously reported that the -866 G/A variation of UCP2 gene was associated with UCP2 mRNA expression in the intraperitoneal adipose tissue. Subjects with G allele of -866 G/A UCP2 gene polymorphism had lower UCP2 mRNA expression compared to those with the A allele [11]. Because UCP2 plays an important role in energy metabolism [9–11], consequently reduction of the expression might affect energy expenditure and this has been confirmed in humans. Kovacs et al. [13] reported that GG genotype of -866 G/A UCP2 gene was associated lower energy expenditure compared to other genotypes (recessive model).
In this study, we found that subjects in GG genotype of -866 G/A UCP2 had higher weight loss when undertaking IFD compared to LCD-ex although subjects in both groups had a comparable amount of dietary intake. The IFD is a new weight loss method that has been just recently introduced in the nutrition community, although it has been practiced for centuries. Studies in animals and humans showed that this diet had an effective weight loss effect as well as improves the metabolic profile [22]. The term of IFD is commonly used when individual periodically abstains from eating for a period longer than an overnight fast and is typically accompanied by a reduction in energy consumption. To date, the IFD could be grouped into 3 categories: alternate day fasting, whole-day fasting and time restricted feeding (TRF). The time restricted feeding is the type of fasting that was being used in this study [22].
One of the biggest obstacles to weight loss is reduction in energy expenditure due to calorie restriction and weight loss and this phenomenon has been well documented in the past decade [23–25]. By contrast, some studies indicated that during the Ramadan fasting period, there were no significant changes in energy production and energy expenditure, although those individuals experienced weight reduction especially from fat loss [26, 27]. This was also supported by another study which showed that when time restriction diet was conducted for 8 weeks, the basal metabolic rate remained unchanged even after subjects had a reduction in BMI [28]. To date, there is no clear explanation on how intermittent fasting was able to reduce body weight and body fat without altering energy metabolism. This mechanism might explain the reason for overweight/obese subjects with GG genotype reduced more weight during IFD compared to LCD-ex.
Another explanation on how individuals with GG genotype of -866 G/A UCP2 responded better during IFD compared to LCD-ex was because of changes in adiponectin. Adiponectin is a protein produced by adipose tissue with the ability to regulate inflammation and energy metabolism [29, 30]. The importance of adiponectin on body weight regulation was proven by the previous investigations which showed that adiponectin concentration in overweight/obese individuals was reduced [31]. It has also been shown that adiponectin treatment was able to induce UCP2 expression and its protein production in animal trials [32] and the positive correlation between adiponectin and UCP2 has been shown in humans[33]. Interestingly, several investigations reported that intermittent fasting was associated with increased adiponectin production [28, 35]. Therefore, we speculated that the increasing adiponectin due to intermittent fasting was able to compensate for lower UCP2 production in GG genotype and thus resulting in weight loss response.
There were several limitations in this study. First, the proportion of women was higher than men in both genotype groups 81% in GG genotype and 66% in AA+GA genotype. However, gender was not significantly associated with differences in percent weight loss in all subjects of UCP2 gene specific groups. Second, this study only showed the short-term effect (4 weeks) because the length of the study was matched to the length of the Ramadan fasting period (1 month). Because treatment of obesity usually requires a longer term of lifestyle changes, further study is needed to confirm whether the effect of IFD on weight loss in GG genotype can be sustained. Third, because of the limitations of budget and facility constraints, physical activity was measured using GPAQ. Although GPAQ has been used worldwide to assess physical activity in population based studies, there might be a bias due to subjective measurement.
In summary, this study supports previous findings which showed that GG genotype of -866 G/A UCP2 (rs659366) gene variation was associated with lower response towards a conventional calorie restriction diet. We found that subjects with GG genotype had a greater weight loss during intermittent fasting diet compared to a conventional low-calorie diet with exercise. Results from this study could be used as a basis for the development of personalized weight loss program based on UCP2 gene variations. Since the current study was conducted in a short period of time, further study is needed to evaluate the long-term impact of this dietary regime.
Conflict of interest and funding disclosure
All authors declare that they have no competing interests. This study was funded by Hibah Penelitian Dana Masyarakat, Fakultas Kedokteran, Universitas Gadjah Mada; Hibah Penelitian Dosen Muda, Universitas Gadjah Mada and Hibah Penelitian Dosen Gizi, Universitas Gadjah Mada.
Individual contribution
Conceptualization and Methodology: H.F.L.M.; Trial and data collection: M.N.H and SAP, Resources, H.F.L.M. Data Curation, H.F.L.M., Writing – Original Draft Preparation, H.F.L.M.; Writing – Review & Editing, H.F.L.M., M.N.H and SAP; Project Administration, M.N.H and SAP; Funding Acquisition, H.F.L.M. Harry Freitag Luglio Muhammad (H.F.L.M.), Satwika Arya Pratama (S.A.P) and Maya Nurfitriani Hartono (M.N.H.).
Footnotes
Acknowledgments
We thank to Fatikhat Nur Latifah, Very Kusumawati, Nur Laila Apriliana, Syari Ernawati Putri, Ayu Larasati, Ika Riski Muharomin for their help during data collection and Dr. Datu Respatika for initial health examination of the participants.