Introduction: Apelin could be one of the last protective defenses before developing obesity-related disorders, including insulin resistance, type 2 diabetes, and hypertension, which can be modified by dietary intake. The present study investigated the association of habitual intake of total fatty acids (TFAs), saturated-, monounsaturated-, polyunsaturated FAs, n-3, and n-6 FAs with Apelin expression in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT). Methods: We obtained VAT and SAT from 168 participants (64 nonobese and 104 obese) who had undergone open abdominal surgery. Dietary intake information was gathered with a valid and reliable food frequency questionnaire. The mRNA expression of the Apelin gene was analyzed by real-time PCR. Results: Apelin serum levels were increased in the obese subjects compared to the nonobese group (p = 0.016). The SAT and VAT Apelin mRNA levels were significantly elevated in the obese participants compared to the nonobese ones (p < 0.05). Based on BMI status, only obese subjects indicated a positive association between SAT and VAT Apelin expression and TFA intake (p < 0.001). However, this association was observed between SAT and VAT Apelin gene expression and polyunsaturated fatty acid (PUFA) and n-3 FA intakes in both obese and nonobese groups (p < 0.05). Conclusion: High Apelin gene expression was associated with TFA intake in obese subjects in both fat tissues. However, habitual intake of PUFA and n-3 FA was associated with Apelin gene expression in obese and nonobese individuals. Our results indicate a determinative role of the quality and quantity of FA intake on adipose tissue.

© 2022 The Author(s). Published by S. Karger AG, Basel

Introduction

Obesity, a major public health problem, is considered as one of the important risk factors for the incidence of metabolic syndrome, atherosclerosis, insulin resistance, dyslipidemia, and type 2 diabetes mellitus [1, 2]. During the last decades, the prevalence of obesity has risen dramatically in many countries throughout the world, which is attributed to changes in lifestyle [3].

Accumulation of excess fat induced by obesity may affect the metabolism of glucose and lipids. It is well known that adipose tissue not only is the largest site reservoir for fats but also acts as an endocrine organ producing and secreting a variety of adipokines that induce insulin sensitivity both locally and in other organs [4].

The human Apelin, an endogenous ligand of APJ, which is a G-protein-coupled receptor, is localized on the chromosome Xq25–26.1 [5]. The Apelin/APJ pathway has several physiological roles, including glucose and lipid metabolism, cardiovascular activity, cell growth, apoptosis, fluid homeostasis, and immune response [6]. To date, a limited number of studies have focused on the role of Apelin in lipid metabolism. In both differentiated 3T3-L1 adipocytes and isolated adipocytes, Apelin inhibits isoproterenol-induced lipolysis via a pathway including Gq, Gi, and AMP-activated protein kinase [7]. Apelin reduces the release of free fatty acid from 3T3-L1 adipocytes by accumulative amount of peripheral lipoproteins surrounding lipoproteins. However, Apelin has no effect on basal or isoproterenol-stimulated lipolysis in human adipose tissue specimen or isolated adipocytes [8]. In standard and high-fat diet-fed mice, daily IP injection of Apelin for 2 weeks has been found to diminish triglyceride (TG) level and fat deposition in adipose tissue at different sites, including a significant decrease in liver TG content and expressions of different adipogenesis-related genes. It was independent of food intake in mice [9]. Among energy-supplying nutrients, FA intakes can modify adipocyte metabolic responses [10-12]. Notably, in a recent systematic review, we described that despite the controversy existing in the findings of related studies, Apelin concentrations might be decreased by hypocaloric diets with a low-fat content in humans. On the other hand, in animals, the consumption of diets rich in fat can result in increased Apelin gene expression in adipose tissue and plasma concentrations [13-15]. The same results were also observed when a standard diet was supplemented with a specific dose of eicosapentaenoic acid (EPA) [16]. Here, we aimed to investigate the association of dietary intake of FAs (total fatty acids (TFAs), saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), n-3 and n-6 fatty acids) with Apelin expression in visceral and subcutaneous adipose tissues (VAT and SAT) among nonobese and obese adults without diabetes.

Materials and MethodsSubjects

For the current cross-sectional study, we selected 168 participants, including 64 nonobese (18.5< body mass index [BMI] <30 kg/m2) and 104 obese (BMI ≥30 kg/m2), who were admitted for elective abdominal surgery at two Khatam al-Anbiya and Mustafa Khomeini hospitals in Tehran, Iran, between 2012 and 2015. We selected individuals who were hospitalized less than 3 days, free of diabetes mellitus or cancer, not taking lipid-lowering drugs, not pregnant or lactating women, and not on special diets. VAT and SAT were collected during the surgery (approximately 100 mg). All questionnaires and other measures were completed before surgery.

Anthropometric and Laboratory Measurements

Weight and height were measured with the precision of 0.1 kg and 0.1 cm, respectively. BMI was calculated as the weight in kilograms divided by the square of the height into meters (kg/m2). Waist circumference was measured at the level of the umbilicus over light clothing using unstretched tape with no pressure to the body’s surface; measurements were recorded to the nearest 0.1 cm. Systolic and diastolic blood pressures were measured twice in the sitting position after at least 10 min of rest, and the average was considered.

Before the surgery, 5 mL of blood (after 10–12 h fasting) was obtained from all participants. To measure fasting plasma glucose (FPG), the glucose oxidase method was applied. Enzymatic methods measured TG and total cholesterol (TC). Intra- and inter-assay coefficients of variation were <1% and <2.1%, respectively. Commercial kits, Pars Azmoon Co. (Tehran, Iran), were used to measure FPG, TG, and TC. Furthermore, the ELISA method determined the insulin level using Mercodia kits (Uppsala, Sweden). The ELISA assay kit determined the Apelin plasma levels (ZellBio, Ulm, Germany). Inter- and intra-assay CVs were 1.7–2.3 and 1.9% for both, respectively.

Dietary Measurements

A valid and reliable semi-quantitative food frequency questionnaire (FFQ) was used, and an expert during face-to-face interviews assessed habitual dietary intake. A trained dietitian asked participants to determine the frequency of each food item that was consumed during the previous year on a daily, weekly, and monthly basis. Overall, the FFQ provides reasonably valid measures of the average long-term dietary intake [17]. FAs and other micro- and micronutrients were calculated from the United States Department of Agriculture (USDA) FCT, and for traditional foods, Iranian FCT was used [18, 19]. The current study considered dietary TFA and its subtypes that included SFA, PUFA, MUFA, n-3, and n-6 FA.

Quantitative Reverse Transcriptase Real-Time Polymerase Chain Reaction

Total RNA was extracted using TRIzol reagent (Invitrogen, US), according to the recommended protocol. The quality of the extracted RNA was evaluated by the NanoDrop spectrophotometer (ND-1000, USA), and the ratios of A260/A280 and A260/A230 of 1.8–2.0 and 2.0–2.2, respectively, were considered as pure. Total RNA (1 μg) was reverse transcribed using cDNA synthetize kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol and stored at −20°C for further use. To evaluate the Apelin gene expression, qRT-PCR was performed by Rotor-Gene 6,000 instrument (Corbett Research, Sydney, Australia). The PCR was completed using the following thermal programs: initial denaturation (5 min at 95°C) and then a three-step amplification program (10 s at 95°C followed by 25 s at 62°C and 35 s at 72°C), with the melting curve, repeated 40 times. GAPDH was used as the reference gene to normalize mRNA levels, based on previously published studies [20]. All experiments were repeated twice. Sequences of the primers are shown in Table 1. PCR amplification was performed in 25 µL volumes using SYBR Green Master Mix (BioFact, Korea). Quantitation of Apelin mRNA level in adipose tissues was evaluated by the comparative 2−∆Ct method, according to Livak et al. [21].

Table 1.Statistical Analysis

We used G*Power to determine sample sizes when the minimum predicted effect size was 0.3 in Pearson correlation, with a two-sided significance threshold set at 5% and power at 95% in addition to 1.1 design effect [22]. For the purpose of the present research, a total of 152 people calculated the minimum needed sample. The normal distribution of variables was checked by Kolmogorov-Smirnov tests and histogram with the normal curve. Continuous variables are described as mean ± standard deviation (SD) or median (interquartile 25, 75%) based on the distributions of variables. T test and Mann-Whitney test were used to compare mean and median of normal distributed and skewed variables in two BMI status groups, respectively. Total dietary fat intake and its subtypes were energy-adjusted by the residual method [23]. To determine the association of energy-adjusted TFA and its subtypes with Apelin gene expression in SAT and VAT, linear regression was performed, adjusted for age and sex. Analyses were performed using Statistical Package for the Social Sciences program (SPSS) (version 15.0; SPSS Inc, Chicago IL, USA), and the null hypothesis was rejected in each statistical test when p value < 0.05.

Results

Anthropometric and laboratory information of participants in two groups of nonobese and obese is provided in Table 2. The mean age of the total sample (n = 168) was 41.4 (min 18 and max 84) years, and 69.6% were women; the mean ± SD of BMI was 24.7 ± 2.82 and 43.1 ± 6.11 kg/m2 for nonobese and obese subjects, respectively (p < 0.001). Apelin serum levels were increased in the obese subjects compared to nonobese group (p = 0.016). Significant differences were observed between groups in all studied variables (p < 0.05), except for age (p = 0.102).

Table 2.

General characteristics (anthropometric and laboratory data) of the study participants

The SAT and VAT Apelin mRNA levels were significantly elevated in the obese participants compared to the nonobese ones (p = 0.030 and p < 0.001, respectively) (Fig. 1). Dietary intakes of participants are provided in Table 3. Mean TFA intake was 31.6% of total energy intake (107 g/d) in the total population. The mean total energy intake was higher in obese individuals than nonobese ones (p < 0.001). Obese subjects also had significantly higher TFA, n-3 FA, and n-6 FA intakes and lower consumption of carbohydrates than nonobese counterparts (p < 0.05). There is no significant association between dietary FA composition and serum concentration of Apelin in obese and nonobese individuals (Table 4).

Table 3.

Dietary intakes of the nonobese and obese participants

Table 4.

Regression coefficients of TFAs and its subtype intake with apelin serum levels in nonobese and obese participants after adjustment for age and sex

Fig. 1.

Apelin gene expression in subcutaneous and visceral adipose tissues in nonobese (BMI <30 kg/m2) and obese (BMI ≥30 kg/m2) subjects; error bars were defined as 1 SEM of the mean.

Associations of SAT and VAT Apelin gene expression with energy-adjusted TFA and its subtypes (residual method) after controlling for age and sex are presented in Table 5. Based on BMI status, only obese subjects indicated a positive association between VAT (β = 0.579, p < 0.001) and SAT (β = 0.658, p < 0.001) Apelin mRNA level and TFA intake. In nonobese participants, PUFA intake was positively associated with Apelin gene expression in VAT (β = 0.424, p = 0.021) and SAT (β = 0.512, p = 0.004). Besides, in obese subjects, it showed a positive association with Apelin gene expression in VAT (β = 0.394, p = 0.024) and SAT (β = 0.407, p = 0.024). Nonobese subjects indicated a positive association between VAT (β = 0.523, p = 0.005) and SAT (β = 0.500, p = 0.004) Apelin mRNA level and n-3 fatty acid intake. As well, obese groups indicated this positive association between VAT (β = 0.558, p < 0.001) and SAT (β = 0.607, p < 0.001) Apelin mRNA level and n-3 fatty acid intake.

Table 5.

Regression coefficients of TFAs and its subtype intake with Apelin mRNA level in nonobese and obese participants after adjustment for age and sex

Discussion

This study showed that habitual dietary intakes of TFA and PUFA were positively associated with Apelin gene expression in VAT and SAT among obese participants. Findings revealed that dietary intake of n-3 FA was positively associated with Apelin mRNA levels in VAT and SAT of both nonobese and obese groups.

It should be noted that the present study was the first investigation that assessed the relationship between habitual intake of FA and Apelin mRNA expression in humans. Previous data on the relationship between TFA and Apelin gene expression in adipose tissue were limited to findings in rodents [13, 24]; generally, Apelin expression in adipose tissues and Apelin concentrations increased in the rodents on a high-fat diet, compared with those on a standard diet [13, 24]. Although animal studies show that Apelin mRNA levels in adipose tissue and serum concentrations after a high-fat diet increased simultaneously [14, 25], another study demonstrated that despite the increase in Apelin gene expression in adipose tissues in response to the high-fat diet, Apelin concentration did not change significantly [15]. Moreover, despite human studies investigating the impact of a low-calorie diet on Apelin concentration among obese patients, no information regarding dietary components was reported [26-28]; hence, we were unable to determine the relation of dietary components especially dietary intakes of FA on Apelin serum levels. In the current study, we observed that TFA was associated with Apelin mRNA levels in the obese subjects’ adipose tissue after controlling for age and sex, thus suggesting that the amount of body fat was not a mediator for an increase in Apelin expression.

We found a significant increase in Apelin mRNA levels and serum levels in the obese subjects compared to the nonobese individuals, which was previously reported [26, 27, 29]. Celik et al. [30] reported that by a 1.12% reduction and Heinonen et al. [28] by a 14.28% reduction BMI no significant change in Apelin serum levels was observed. Therefore, in addition to weight status, some other related factors might also modify the concentration of Apelin. Insulin level is a potential factor in Apelin changes. Decreased insulin concentration has possibly more influence than merely weight loss. Therefore, insulin levels may have a mediated role in the existing direct relationship between Apelin and excess weight [13, 14, 24, 31, 32]. Participants who were free of diabetes and hypertension were recruited for the present study. Furthermore, the nutrient content of the habitual diet may be among other factors that influence Apelin. In the current study, high intakes of PUFA and n-3 FA predicted Apelin gene expression in both SAT and VAT in two studied BMI groups.

Apelin expression in response to EPA intervention was investigated in adipose tissues and skeletal muscle [13, 14]; EPA supplementation (1 g/kg) in rats with a high-fat diet increased Apelin gene expression [14]. Moreover, in mice that received a high-fat diet enriched with 36 g/kg EPA, adipose tissue Apelin gene expression was increased [13]. In this study, supplementation with EPA also led to reduced levels of insulin and glucose as well as improved insulin sensitivity through the increment of β oxidation in insulin-dependent organs [13]; similarly, Apelin was also demonstrated to enhance glucose tolerance in mice with obesity and insulin resistance [33]. In addition, the present study showed n-3 FA predicted Apelin gene expression of a similar amount in both adipose tissues in obese subjects and 1-SD increase in n-3 FA had an association with approximately 0.6 unit increases in both SAT and VAT Apelin mRNA levels. In addition, it seems that n-3 FA intakes increased Apelin gene expression more in SAT among obese subjects than in nonobese ones (0.7 vs. 0.5). Perez-Echarri et al. [14] showed that in rats, overfeeding with a diet rich in SFA increased Apelin gene expression in visceral fat. However, Apelin gene expression in rats fed with a high-SFA diet along with EPA treatment was higher than that fed to rats, i.e., high-fat diet [14].

The potential mechanism of the effect of dietary FA intake on Apelin is not well understood; however, some mediator pathways can be suggested. Insulin and leptin are potential mediators for regulating the diet-induced Apelin levels [13, 34-38]. In addition to indirect stimulation, previous research in cultured adipocytes suggests that the stimulation of Apelin gene expression seen following EPA supplementation in vivo is likely related to EPA’s capacity to directly alter the processes involved in adipocyte Apelin production and secretion [39].

The overexpression and activation of peroxisome proliferator-activated receptor-γ (PPARγ) is one possible mechanism for n-3 fatty acid’s influence on increasing Apelin gene expression from adipocytes. High levels of PPARγ expression and activation boost Apelin production [40]. In vitro, higher fatty acids, especially PUFA, were shown to bind directly with PPARγ at amounts similar to those consumed and absorbed by humans, suggesting that they may operate as natural ligands for this receptor [41].

There were limitations that should be stated. Small sample sizes limited our power to detect statistical associations. Despite the relatively large magnitude of the β standard in the dietary exposure and Apelin mRNA levels, the exploratory insight of the current study should be considered. Therefore, our results need to be confirmed in the cohort and trial studies. Because the current study’s design was cross-sectional, a causal relationship cannot be inferred. However, since it is less probable that Apelin induces dietary fat quality, we assumed that our conclusion is likely that dietary fat may impact Apelin gene expression in adipose tissue. The nonrandom sample used in this research may have also resulted in a selection bias, so caution should be used when interpreting the findings. Convenience sampling may prevent results from being generalized to the whole population and gradually losing their representativeness of the target group. The results of subsequent research must take sample recruitment into account in order to be comparable and generalizable. Although there is a chance of recall bias given that the FFQ application covers a broad range of foods consumed over the course of a year, we minimize the bias by application of the validated and reliable FFQ and completing the FFQ during individual face-to-face interviews conducted by expert dietitians in this study reduces this type of error. Moreover, another limitation was the lack of measuring Apelin protein concentration and APJ gene expression in adipose tissue.

Conclusion

Our results demonstrate a strong relationship between dietary intakes of PUFA and n-3 FA and Apelin mRNA levels in adipose tissues of obese and nonobese groups, which indicate the importance of FAs’ quality in addition to quantity in a diet in terms of the Apelin gene expression in adipose tissues. These significant findings provide a good starting point for further research in understanding the mechanism underlying Apelin gene expression and FAs metabolism.

Statement of Ethics

Ethics approval was obtained from the Research Institute for Endocrine Sciences (RIES) Ethics Committee of the Shahid Beheshti University of Medical Sciences (NO: IR.SBMU.ENDOCRINE.REC.1395.169) and conducted under the Declaration of Helsinki as well as our institutional guidelines. Written informed consent was obtained from all participants.

Conflict of Interest Statement

The authors declare that they have no competing interests.

Funding Sources

There was no special funding to do this project.

Author Contributions

Maryam Zarkesh conceptualized and designed the study, gathered adipose tissues, prepared the manuscript, and approved the final manuscript as submitted; Emad Yuzbashian analyzed and interpreted the data, prepared the manuscript, and approved the final manuscript as submitted; Afsoon Daneshafrooz prepared the laboratory materials, cDNA synthesized, RNA extraction, performed real-time PCR, and approved the final manuscript as submitted; Golaleh Asghari entered data, drafted the initial manuscript, and approved the final manuscript as submitted; Mehdi Hedayati supervised the project, consulted laboratory protocol, and approved the final manuscript as submitted; Parvin Mirmiran drafted the initial manuscript and approved the final manuscript as submitted; Alireza Khalaj biopsied the patients during the abdominal surgery and approved the final manuscript as submitted; Mohammad Safarian supervised the project and approved the final version of the manuscript as submitted.

Data Availability Statement

The datasets analyzed during the current study are available from the corresponding author on reasonable request

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