**1. Introduction**

The pandemic of obesity has been termed "globesity" to remark the global nature of the problem. Lifestyle modifications with a low-calorie diet produce weight loss and the secondary improvement of many of the components of associated comorbidities, including hyperlipidemia, diabetes mellitus type 2, hypertension, and inflammatory markers [1]. Now, adipose tissue has been considered an important cornerstone endocrine organ secreting several adipokines implied in the regulation of metabolism and energy status. Some adipokines have a proinflammatory role as leptin and resistin and other groups such as adiponectin has an anti inflammatory function [2]. Adiponectin is the most important adipokine secreted by this tissue [3]. Adiponectin has an anti-inflammatory role and its levels are reduced in obese subjects and are enhanced after weight reduction [4]. Low adiponectin levels have been related with a high risk of obesity, diabetes mellitus, and hyperlipidemia [5], with a potential therapeutic effect with agonists of adiponectin [6].

The adiponectin levels are highly heritable, and the *ADIPOQ* gene is the principal locus promoting variations in serum levels [7]. Single nucleotide polymorphisms (SNPs)

**Citation:** de Luis Roman, D.A.; Primo, D.; IZaola, O.; Gómez, E.; López, J.J. Adiponectin Gene Variant rs3774261, Effects on Lipid Profile and Adiponectin Levels after a High Polyunsaturated Fat Hypocaloric Diet with Mediterranean Pattern. *Nutrients* **2021**, *13*, 1811. https:// doi.org/10.3390/nu13061811

Academic Editor: Martina Barchitta

Received: 8 May 2021 Accepted: 24 May 2021 Published: 26 May 2021

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are genetic variants that can sometimes have functional implications in the *ADIPOQ* gene, which is situated on chromosome 3q27. One of these SNPs, 712 G/A rs3774261 in the *ADIPOQ* has been related to diabetes mellitus in obese subjects [7] and with coronary heart disease [8,9]. Interestingly, this genetic variant has been associated with eating behavior [10], and there are nutritional intervention studies [11,12], too. These interventional designs [11,12] reported significant results on serum lipid profile and inflammatory markers. Our previous study with a Mediterranean diet [11] had a lower percentage of fat in the diet than the current intervention and low sample size; with this new study, we will evaluate more precisely the effect of the quantity and quality of fat in a larger sample of patients. Perhaps the metabolic effects found in these studies and their relationship with this genetic variation are due to both factors; weight loss and the Mediterranean diet pattern used. The beneficial effects of a diet with a Mediterranean style can be due to the presence of different foods and nutrients such as type of dietary unsaturated fatty acids [13]. Unsaturated fatty acids are ligands for the transcription factor PPAR gamma [14], which increases *ADIPOQ* gene expression and improves adiponectin concentration [15]. Perhaps increasing the amount of unsaturated fat in a hypocaloric diet would have greater benefits than a conventional hypocaloric diet and rs3774261 would modulate these changes. In previous studies [11,12], the beneficial effects found with the Mediterranean diet were related to the high consumption of olive oil, and therefore, of monounsaturated fatty acids. Notwithstanding that the polyunsaturated fatty acids may also play a relevant role and interact with this genetic variant of the ADIPOQ gene, this hypothesis has not ye<sup>t</sup> been evaluated.

Given this lack of information, we conducted a study to evaluate the effects of a high polyunsaturated fat hypocaloric diet with a Mediterranean style during 12 weeks on metabolic changes considering the rs3774261 of *ADIPOQ*.

### **2. Subjects and Methods**

### *2.1. Subjects and Clinical Investigation*

Obese subjects were enrolled by the primary care physicians of our health area to treat obesity. These subjects were evaluated in a single-arm clinical trial with a high fat polyunsaturated hypocaloric diet with a dietary Mediterranean pattern. The local ethics committee (Hospital Clinico Universitario Valladolid committee 7/2017, code: GRS588/A/11) approved the protocol; it was in accordance with the guidelines laid down in the Declaration of Helsinki and all subjects gave written informed consent. Clinical and biochemical variables were recorded at the beginning and after 12 weeks of dietary interventions. All the enrolled obese subjects met the following inclusion criteria; age between 30 and 60 years old and an obesity category as a body mass index (BMI) ≥ 30 kg/m2. The exclusion criteria were any of the following data: previous cardiovascular event, chronic renal failure, chronic liver failure, alcoholism, malignant tumor, and within the 24 weeks before the study were taking any medications or nutrient-supplements or have been on a low-calorie diet.

The main objective of our study was serum adiponectin change after 12 weeks versus baseline. The secondary objectives were improvements in lipid profile and glucose metabolism after dietary intervention. Lipid profile (LDL-cholesterol, HDL-cholesterol, triglycerides, and total cholesterol), C-reactive protein (CRP), insulin, and adipokines (resistin leptin and total adiponectin) levels were analyzed. Homeostasis model assessment (HOMA-IR) and adiponectin/leptin ratio were calculated, too. The anthropometric evaluation was realized with body weight, height, waist circumference, fat mass by bioimpedance, and calculated body mass index (BMI). Systolic and diastolic blood pressure was recorded. All clinical and biochemical parameters were determined at the basal time and after 3 months of dietary intervention. The genetic variant rs3774261 of the *ADIPOQ* gene was assessed.

### *2.2. Dietary Intervention*

A total of 361 obese patients met the above-mentioned criteria, and they were included to observe a hypocaloric diet for 12 weeks. The diet (high-polyunsaturated fatty acid hypocaloric diet Mediterranean diet) was based on a calorie restriction of 500 calories over the usual intake, 45.7% of carbohydrates, 34.4% of fats, and 19.9% of proteins. The percentages of different fats were: 21.8% of monounsaturated fats, 55.5% of saturated fats, and 22.7% of polyunsaturated fats (13 g per day of w-6 fatty acids, 3 g per day of w-3 fatty acids, and a ratio w6/w3 of 4.3).

Food tables were used with a Mediterranean dietary style, including (legumes, vegetables, and fresh fruit 5 servings per day, poultry, whole grains, fish 3 times per week, using 20 g olive oil per day, 40 g of walnuts daily, and limit unhealthy fats such as margarine, fatty meats, snacks, industrial pastries) [16]. To improve compliance with dietary intervention, the completion of diet recommendations was evaluated every 10 days with a phone call. Records of daily dietary intake for 4 days were parameterized with software (Dietosource®, Geneva, Switzerland), and this software is based on national composition food tables [15]. The recommendations for physical activity were aerobic physical activities at least 3 times each week (60 min each). The physical activity allowed by protocol were (cycling, running, walking, and swimming). Each patient with a self-reported questionnaire recorded the physical activity.

### *2.3. Biochemical Parameters*

Blood samples were drawn after a minimum of 10 h overnight and these samples were stored at −80 ◦C until analyzed. Lipid profile (low-density lipoprotein (LDL)-cholesterol, high-density lipoprotein (HDL)-cholesterol, triglycerides, and total cholesterol), C reactive protein (CRP), fasting glucose, and insulin levels were determined on the same day using the clinical chemistry automated analyzer COBAS INTEGRA 400 analyzer (Roche Diagnostic, Montreal, Canada). LDL cholesterol was calculated using the Friedewald formula (LDL cholesterol = total cholesterol-HDL cholesterol-triglycerides/5) [17]. Insulin resistance was calculated using the homeostasis model assessment (HOMA-IR) with the following equation (glucosexinsulin/22.5) [18].

Serum adipokines were measured by enzyme-linked immunosorbent assays (ELISA). Resistin kit had with a normal range of 4–12 ng/mL [19] (Biovendor Laboratory, Inc., Brno, Czech Republic). The leptin kit had a normal range of 10–100 ng/mL (Diagnostic Systems Laboratories, Inc., Webster, TX, USA) [20]. Finally, the adiponectin kit had a normal range of 8.65–21.43 ng/mL (R&D Systems, Inc., Minneapolis, MN, USA) [21]. Adiponectin/leptin ratio was calculated in all samples.

### *2.4. Genotyping ADIPOQ Gene*

The genotype of SNP rs3774261 of *ADIPOQ* was determined with a polymerase chain reaction in real-time from peripheral blood leucocytes. Genomic DNA was obtained from a 150 uL buffy coat using a blood genomic kit (Bio-Rad®, Hercules, CA, USA) in accordance with the manufacturer´s instructions. Probes and oligonucleotide primers and were designed with the Beacon Designer 5.0 (Premier Biosoft International®, LA, CA, USA). The polymorphic region of adiponectin was amplified using the polymerase chain reaction (PCR) with 50 ng of this genomic DNA, with allele-specific sense primers (primer forward: 5'-ACGTTGGATGCTCCTCCTTGAAGCCTTCAT-3' and reverse 5'-ACGTTGGATGCAAGTATTCAAAGTATGGAGC-3' in a 2 μL final volume (Termocicler Life Technologies, LA, CA, USA). Cycling parameters were as follows: after DNA denaturation at 95 ◦C for 1 min and annealing at 65 ◦C for 30 s. The PCR was run in a 25 μL final volume containing 10.5 μL of IQTM Supermix (Bio-Rad®, Hercules, CA, USA) with hot start Taq DNA polymerase. Duplicates in the arrays were the methodology to internal controls and the accuracy. Hardy Weinberg equilibrium was determined with a statistical test (Chi-square) to compare our expected and observed counts. The variant was in Hardy Weinberg equilibrium (*p* = 0.31).

### *2.5. Anthropometric Parameters and Blood Pressure*

Bodyweight, height, and waist circumference (WC) were determined in the morning before breakfast at baseline and after 3 months. Body mass index was determined by the equation (weight in kg divided by the height in meters squared). Bodyweight was determined with a scale (Omron, LA, CA, USA), and the obese subjects were minimally unclothed to the nearest 0.1 kg and not wearing shoes (Omron, LA, CA, USA). Fat mass was estimated by bioimpedance (Akern, EFG, Pisa, Italy) with an accuracy of 50 g [22]. WC was measured with a measuring tape in the narrowest diameter between the xiphoid process and iliac crest. Systolic and diastolic blood pressures were measured three times and averaged after a 5 min rest with a random zero mercury sphygmomanometer, (Omron, LA, CA, USA).

### *2.6. Statistical Analysis*

We used the software SPSS for Windows, version 23.0 software package (SPSS Inc. Chicago, IL, USA) to analyze the data. The sample size was determined to assess changes over 5 ng/mL of adiponectin levels with 90% power and 5% significance (*n* = 300). Results were expressed as average ± standard deviation. Each variable was evaluated for normality with the Kolmogorov–Smirnov test. The parametric test was investigated with the ANOVA test and Bonferroni post hoc test. Non-parametric parameters were evaluated with the Mann-Whitney U-test. Categorical variables were revised with the chi-square test, with Yates correction as necessary, and Fisher's test. The gene–diet interaction was assessed with a univariate ANCOVA adjusted by gender, and baseline weight. Correction for multiple hypotheses testing for single SNP analyses was performed. A Chi-square test was used to determine the Hardy–Weinberg equilibrium. A *p*-value < 0.05 was considered significant.

### *2.7. Ethical Approval*

All methodology of our study were in accordance with the ethical standards of the institutional and/or national research committee (Hospital Clinico Universitario Valladolid committee 7/2017, code: GRS588/A/11) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was signed from all individual participants included in the study.
