**3. Results**

### *3.1. Characteristics of Participants and Dietary Intakes*

We evaluated the role of SNP rs3774261 on the modification of adiposity markers and biochemical variables in 361 obese outpatients. The average age of the sample was 47.1 ± 3.1 years (range: 29–63) and the average body mass index (BMI) was 37.3 ± 4.9 kg/m<sup>2</sup> (range: 33.5–49.9). Sex distribution was 259 females (71.7%) and 102 males (28.3%). The genotype distribution of this sample was as follows: 117 patients (32.4%) AA, 164 patients AG (45.4%), and 80 patients GG (22.2%). Allelic frequency was 0.62 A and 0.38 G. Sex distribution and the average age was similar in all genotype groups (Table 1).

Following the sessions of the dietitian, the dietary recommendations were reached at 12 weeks in all genotype groups with a significant decrease of total caloric amount, carbohydrates, fats and proteins (Table 1). A significant increase was observed in the percentage of monounsaturated and polyunsaturated fats (Table 1).

At basal time, the physical activity was similar in the three groups (Table 1). In addition, after the intervention, the physical activity improved, but this improvement did not show differences in total quantity deltas (AA vs. AG vs. GG) (28.2 ± 1.2 min/week vs. 29.8 ± 2.1 min/week vs. 29.1 ± 1.1 min/week; *p* = 0.52).


**Table 1.** Changes in anthropometric parameters, dietary intakes, and physical activity rs3774261 (mean ± S.D).


**Table 1.** *Count*.

BMI: body mass index. SB: Systolic blood pressure. DB: Diastolic blood pressure WC: Waist circumference. (\*) *p* < 0.05, in each genotype group. No differences between genotype groups.

### *3.2. Anthropometric Results*

For rs3774261, there were no statistical differences in anthropometric parameters and systolic/diastolic blood pressure in basal and post-intervention values (AA vs. AG vs. GG) (Table 1). After a high polyunsaturated fat hypocaloric diet with a Mediterranean style, we observed a significant improvement of body mass index, weight, waist circumference, fat mass, and systolic blood pressure. These statistically significant changes were similar in all genotype groups. Diastolic blood pressure remained unchanged.

### 3.2.1. Biochemical Parameters

In the second analysis of our design, we evaluated the actions of this dietary intervention on glucose metabolism, C Reactive protein and lipid profile (Table 2). After a significant body weight loss (AA vs. AG vs. GG); insulin levels (delta: −3.7 ± 0.2 UI/L vs. −3.8 ± 0.3 UI/L vs. −3.6 ± 0.2 UI/L; *p* = 0.33) and HOMA-IR (delta: −1.3 ± 0.2 units vs. −1.2 ± 0.3 units vs. −1.1 ± 0.4 units; *p* = 0.36) improved in all genotypes without intergroup differences. Finally, after 12 weeks (AA vs. AG vs. GG); total cholesterol (delta: −28.1 ± 2.1 mg/dL vs. −14.2 ± 4.1 mg/dL vs. −11.0 ± 3.9 mg/dL; *p* = 0.01), LDL cholesterol (delta: −17.1 ± 2.1 mg/dL vs. −6.1 ± 1.9 mg/dL vs. −6.0 ± 2.3 mg/dL; *p* = 0.01), triglyceride levels (delta: −35.0 ± 3.6 mg/dL vs. 10.1 ± 3.2 mg/dL vs. −9.7 ± 3.1 mg/dL; *p* = 0.03) and C reactive protein (CRP) (delta: −2.3 ± 0.1 mg/dL vs. −0.2 ± 0.1 mg/dL vs. −0.2 ± 0.1 mg/dL; *p* = 0.01) improved only in the AA group.

### 3.2.2. Adipokine Levels

Table 3 reports changes on serum adipokines and ratio adiponectin/leptin. After dietary intervention, in the AA genotype group (AA vs. AG vs. GG), serum adiponectin (delta: 11.6 ± 2.9 ng/dL vs. 2.1 ± 1.3 ng/dL vs. 3.3 ± 1.1 ng/dL; *p* = 0.01) improved. Adiponectin/leptin ratio improved in the AA genotype group (delta: 1.5 ± 0.1 ng/dL vs. 0.3 ± 0.2 ng/dL vs. 0.4 ± 0.3 ng/dL; *p* = 0.02). In all genotype groups, leptin decreased in a significant way. Serum resistin levels did not change after the dietary intervention.


**Table 2.** Biochemical parameters rs3774261 (mean ± S.D).

Total Ch: Cholesterol. TG: Triglycerides LDL-ch: Low density lipoprotein cholesterol, HDL-ch: High density lipoprotein cholesterol. CRP: c reactive protein. HOMA-IR: Homeostasis model assessment. LDL: low density lipoprotein, HDL: High density lipoprotein. (\*) *p* < 0.05, in each group. No statistical differences among genotypes in basal time or after 12 weeks. See significant deltas in the text.


**Table 3.** Serum levels of adipocytokines (mean ± S.D).

(\*) *p* < 0.05, in each group with basal values. No statistical differences among genotypes in basal time or after 12 weeks. See significant deltas in the text.

### **4. Discussion**

We revealed, in this study on a high-polyunsaturated fat hypocaloric diet with a Mediterranean dietary pattern, a decline in LDL-cholesterol, triglycerides, and C reactive protein (CRP) and a rise in serum adiponectin levels and adiponectin/leptin ratio that were statistically significant in obese subjects with the AA genotype of rs3774261. In addition, all subjects in all genotype groups showed a significant decline of adiposity parameters and systolic blood pressure after dietary intervention.

In the literature, some investigations have shown the relationship between this genetic variant (rs3774261) on *ADIPOQ* gene and obesity, metabolic syndrome, diabetes mellitus, and serum adiponectin levels [23,24], with an increased risk to present type 2 diabetes, obesity, and hypoadiponectinemia in a non-Caucasian population [24,25]. In addition to this association with high cardiovascular risk pathology, the G allele of rs3774261 *ADIPOQ* has also been related to coronary heart disease [8]. The exact pathways by which genetic variants in the *ADIPOQ* gene produce coronary heart disease is not well known. Moreover, adiponectin, the adipokine encoded by this gene, has been reported to have antiinflammatory properties that have important effects in fighting against atherosclerosis [26].

There is little information in the literature about the effect of nutritional treatment and this genetic variant. A recent study with a normal hypocaloric Mediterranean diet reported [11] better changes in lipid profile, CRP, and adiponectin levels in subjects with AA genotype compared in a dominant model with (AG + GG). This previous study [11] had a small sample size (*n* = 135) and a separate analysis of the three genotypes could not be performed. In another design with 284 Caucasian obese subjects [12] reported that after a high-fat hypocaloric diet, the response of lipid levels, CRP, and adiponectin was better on AA genotype than GA or GG genotypes, as our present results. The amount of lipid profile improvement was similar in both studies [11,12] and our present study; however, the rise in adiponectin levels and the decline in CRP was two times greater in the study with a high-fat diet [12] than in the other one [11]. In addition, these changes in lipids and CRP are of a similar magnitude to those found in the current study with a

high-polyunsaturated fat hypocaloric diet. The caloric restriction was similar in these three studies, about 500 calories to the previous intake. Although both strategies were carried out with a Mediterranean diet pattern, it is important to remark that in the last study [12] and the present study, the percentage of fat in the diet was higher than the old one; 38% [12] and 34% (current) vs. 25% [11]. In our investigation, patients reached a daily intake of almost 30 g of monounsaturated fat and 16 of polyunsaturated fat, compared to 14 g and 3.6 g [11], and 25 g and 13 g [12], respectively.

This relationship of the quality of dietary fats with the biochemical changes due to weight loss and its interaction with this genetic variate of the ADIPOQ gene could be explained with some findings in the literature with other SNPs in this gene. For example, Alsahel et al. [27] showed that the genetic variant (-1006G/A) increased serum adiponectin after a high-monounsaturated diet, whereas in A-allele carriers it is decreased. This differential response was not detected with a low-fat hypocaloric diet. Moreover, the molecular mechanism could be related to the potential action of dietary unsaturated fat as ligands of PPAR gamma. The role of polyunsaturated fatty acids is being evaluated in the literature, and more intervention studies are recommended with known doses of fatty acids to evaluate their effect on adipokines [28]. In addition, this metabolic way could explain our findings on inflammatory status with CRP levels. The NF-kB pathway in the endothelium, which would produce CRP; was inhibited by adiponectin [29], for example, adiponectin contributes more powerfully to CRP elevation than for example smoking habit, age, and other metabolic parameters [29].

It seems clear in the literature that this genetic variance is related to a pro-inflammatory status, not only related to ischemic heart disease [8] but also with ischemic stroke patients [30]. In our interventional design, the response of insulin resistance was similar in all genotypes. Moreover, some studies have reported that patients carrying the A allele have better insulin sensitivity demonstrated by the euglycemic clamp [31]. The response of triglycerides found in our trial may also be important since the association between rs3774261 and coronary heart disease [8] was influenced by interactions with serum triglycerides [8]. We must not forget the differentiated response of adiponectin levels as a function of genotype found in our study to explain our metabolic findings. A novelty finding in our study was the association of this genetic variant with the adiponectin/leptin ratio and its secondary modifications to the diet. The adiponectin/leptin ratio is a biomarker of adipose tissue dysfunction and inflammation [32], which related leptin as an adipokine related to the degree of adiposity [33]. Moreover, an adiponectin/leptin quotient higher than the unit is considered as normal whereas a ratio below or near to 0.5 units may show an increase in the metabolic risk [34], as reported by the three genotypes in our study before weight loss and near to 0.5 in GA and GG genotypes after dietary intervention.

In addition to the relationship between inflammatory pathways and adiponectin response to explain our findings, unknown genetic mechanisms may also be implicated. The rs3774261 variant is in an intron located, a non-coding region. Moreover, the genetic variant in non-coding regions may alter gene splicing, transcription product binding, mRNA deterioration, and gene expression. Thus, any of these above-mentioned mechanisms can be possible pathways to explain our findings. For example, with different levels of adiponectin circulating in a case-control study, a relationship of the G allele of this genetic variant with the prevalence of metabolic syndrome was found [35]. All these relationships are complicated by the existence of a relevant role of adiponectin at the level of the central nervous system in the pathways of hunger and satiety. Recently it has been observed that the rs3774261 variant is related to disinhibition in eating behaviors [10] influencing daily total food uptake.

There are some limitations of this study. First, we only evaluated one SNP of *ADIPOQ*, so other variants could be related to metabolic parameters. Moreover, some synthetic associations of specific unusual variants may be in partial linkage disequilibrium with usual variants as rs3774261. Second, the lack of a control group without diet might be a bias. It might provide more valuable information on the relationship of *ADIPOQ*

*variants* (rs3774261) with metabolic parameters if the comparative study was conducted on an unrelated control population. Third, the self-reported dietary intake of energy and macronutrients is not reliable and it might include bias of under-or over-reporting. Finally, we studied a Caucasian population, and extrapolation to other populations is not possible.
