*3.1. Basic Characteristics Depending on Co*ff*ee Consumption*

Table 2 shows the basic characteristics of the subjects according to sex and the amount of coffee intake. In both male and female subjects, those in the high coffee intake group were younger, had a higher income, had a longer duration of education, and were more frequently current smokers when compared with the findings in the low coffee intake group. Energy consumption was higher in the high coffee intake group than in the low coffee intake group. Additionally, the high coffee intake group showed a significantly lower consumption of sugar and the proportion of carbohydrates in energy distribution when compared with the findings in the low coffee intake group.




**Table 2.** *Cont.*

<sup>1</sup> Data are presented as the means ± SDs or n (%). <sup>2</sup> Statistical significance was calculated with Student's *<sup>t</sup>*-tests for continuous variables after log transformation and chi-square tests for categorical variables. <sup>3</sup> Married included married and cohabitation. <sup>4</sup> Data were collected from current alcohol consumers without missing responders; *n* = 610, 1216 in male and 220, 421 in female, respectively. <sup>5</sup> Data were collected from former and current smokers without missing responders; *n* = 189, 372 in male and 13, 24 in female, respectively. <sup>6</sup> Degree of obesity was categorized into four stages according to the criterion of World Health Organization (WHO) Asia-Pacific Area [21]. <sup>7</sup> Subjects with diagnosis in medical history.

In contrast, the mean intake of fat was higher in the high coffee intake group than in the low coffee intake group, and the finding was in accordance with an increased ratio of energy distribution. Systolic blood pressure was lower in the high coffee intake group than in the low coffee intake group. However, hip circumference, height, and weight were higher in the high coffee intake group than in the low coffee intake group. Among both male and female subjects, the TC level was higher in the high coffee intake group than in the low coffee intake group. However, the TG level was higher among male subjects and lower among female subjects in the high coffee intake group than in the low coffee

intake group. Among female subjects, the prevalence of hypertension and menopause were lower in the high coffee intake group than in the low coffee intake group.

### *3.2. Association of Co*ff*ee Intake with the Risk of Dyslipidemia*

We next examined the effect of coffee intake on dyslipidemia risk. There was an inverse correlation between coffee intake and the prevalence of dyslipidemia in female subjects (OR: 0.768, 95% CI: 0.645–0.914, *p* = 0.0030) but not in male subjects (*p* = 0.2635) after adjusting for confounders (Table 3).


**Table 3.** Associations between coffee intake and the risk of dyslipidemia.

<sup>1</sup> Adjusted for age, marital status, income, education, smoking behavior, energy intake, systolic blood pressure, and BMI. <sup>2</sup> Adjusted for age, income, education, drinking and smoking behavior, energy intake, BMI, menopause, treatment of female hormone, and hypertension. <sup>3</sup> Odds ratio (OR), 95% confidence interval (95% CI), and statistical significance were calculated with logistic regression analysis. DLP, dyslipidemia.

#### *3.3. E*ff*ects of Co*ff*ee Intake on the Risk of Dyslipidemia Depending on ADORA Gene Family*

Finally, we performed a logistic regression analysis to confirm the genetic effect of the *ADORA* gene family on the association between coffee intake and dyslipidemia risk (Tables 4 and 5). Interestingly, among female subjects, a favorable effect of consuming more coffee on dyslipidemia risk showed only those with the minor alleles of *ADORA1* rs10800901 (OR: 0.727, 95% CI: 0.560–0.944, *p* = 0.0168), and *ADORA2B* rs2779212 (OR: 0.645, 95% CI: 0.506–0.823, *p* = 0.0004) and the major alleles of *ADORA3* rs2786967 (OR: 0.818, 95% CI: 0.676–0.989, *p* = 0.0384), but not in those with alternative alleles. Among male subjects, there was instead an increased dyslipidemia risk on consuming more coffee carrying the minor alleles of *ADORA2A* rs57604223 (OR: 1.352, 95% CI: 1.014–1.802, *p* = 0.0402). Male subjects with the minor allele of *ADORA3A* rs3393 also showed lower risk on dyslipidemia (Table S1), and the favorable effects did not occur when they consumed more coffee. Overall, these results indicate that the effect of coffee intake on dyslipidemia risk depends on genetic variants in the *ADORA* gene family in a sex-specific manner.


**Table 4.** Risk of dyslipidemia depending on the coffee intake and genotype in ADORA gene family in male.


**Table 4.** *Cont.*

<sup>1</sup> Adjusted for age, marital status, income, education, smoking behavior, energy intake, systolic blood pressure, and BMI. <sup>2</sup> *p* for interaction.


**Table 5.** Risk of dyslipidemia depending on the coffee intake and genotype in ADORA gene family in female.


**Table 5.** *Cont.*

<sup>1</sup> Adjusted for age, income, education, drinking and smoking behavior, energy intake, BMI, menopause, treatment of female hormone, Hypertension. <sup>2</sup> *p* for interaction.

#### **4. Discussion**

The present study aimed to investigate whether genetic variants in the *ADORA* gene family influence the effect of coffee intake on dyslipidemia risk. Coffee intake was associated with decreased dyslipidemia risk in female subjects but not in male subjects. Furthermore, with regard to the genetic effect on the association, the favorable effect of coffee intake among female subjects depends on a subset of genetic variants in *ADORA* gene family. The risk of dyslipidemia was also increased among male subjects in the high coffee intake group based on genetic variation of the *ADORA* gene family, indicating that a subset of genetic variants in the *ADORA* gene family modulates the effect of coffee intake on dyslipidemia risk in a sex-specific manner.

The *ADORA* gene family has been reported to play a role in regulating the lipid profile [12]. For instance, ADORA1 deficiency in ApoE KO mice was associated with increased plasma lipid levels [22], and *ADORA2B* knockout mice showed increased TG and TC levels compared to the wildtype [23]. Disturbed lipid levels via modulation of *ADORA2B* also influenced the development of dyslipidemia and atherosclerosis, known risk factors of cardiovascular mortality [16]. ADORA2B also showed a close relationship with cholesterol regulation by formation of foam cells and inflammation, which are mediator of cardiovascular disease [13,16]. In addition to the functional relevance of the ADORAs in blood lipid profiles and lipid-related chronic diseases, a genetic variant of *ADORA2A* showed association with the severity of chronic heart failure in Asians [15]. The evidence proposed that variations in the ADORA gene family might influence lipid regulation and cardiovascular disease. We also observed a subset of genetic variants in the *ADORA* gene family associated with the risk of dyslipidemia (Table S1).

Despite the interesting finding of an association between the *ADORA* gene family and dyslipidemia itself, the novelty here is that the ADORAs modulates the effect of coffee intake on dyslipidemia. A meta-analysis showed coffee intake increase blood lipid level [2], but not all of the included studies satisfied the result [3–5]. We identified different effects of coffee intake in the risk of dyslipidemia linked to their genetic variants in the *ADORA* gene family. Even though there was no association between coffee intake and dyslipidemia in male, we confirmed the increased risk of dyslipidemia

when subjects with the minor allele of rs5760423 in *ADORA2A* consumed more than one cup of coffee. While we did not experimentally examine the association, instead only focusing on the association of genetic variants in *ADORA* gene family with coffee intake in dyslipidemia, we identified a subset of genetic variants in the *ADORA* gene family located at regulatory elements which could play a role as eQTLs influencing gene expression in various tissues [24] (Table 1). Indeed, a recent study suggested that genetic variation could contribute to altered gene expression by changing epigenetic enhancer activity, which, in turn, is linked to five different vascular diseases [25]. Given the previous reports, genetic variants in ADORA gene family might modify gene expression through epigenetic regulation, possibly modulating the lipid profile and the effect of coffee intake in dyslipidemia pathogenesis. Further studies are needed to elucidate their possible functional mechanisms.

We also observed a favorable association between coffee intake and the prevalence of dyslipidemia in female subjects but not in male subjects. Inconsistent results of coffee intake between male and female individuals [26,27], including a Korean population [28], obscure the view. Female individuals responded favorably to coffee concerning cardiovascular health. It has been proposed that the female sex hormone estrogen plays a role in the sensitivity of female individuals to the effects of coffee intake [29]. Estrogen is synthesized from cholesterol in the ovary, and it influences lipid metabolism by increasing lipoprotein lipase activity and is directly interacting with specific estrogen receptors in the adipose tissue. Thus, susceptibility to cardiovascular diseases is lower in premenopausal women than in men of the same age and postmenopausal women [30]. A previous finding that coffee intake increases the concentration of estrogen in Asian female individuals could explain the sex-specific differences in the effect of coffee intake on dyslipidemia [29].

The most interesting of our findings is that increased coffee intake had beneficial effects in female subjects but harmful effects in male subjects significantly associated with a subset of genetic variants in the *ADORA* gene family. This could suggest that the response to environmental factors of the ADORAs differs according to sex. Several previous studies showed different influences of environmental factors related to the *ADORA* genotypes depending on sex. Treatment with the ADORA antagonist ATL444 was shown to have a preventive effect on cocaine addiction in male individuals but not in female individuals [31]. Additionally, locomotor activity in response to administration of caffeine was higher in male WT mice than in male *ADORA2A* knockout mice, however, this difference was not noted in female mice. Although the reason why the *ADORA* genotype causes a difference in the environmental response depending on sex is not known, a possible explanation may be that dopamine receptor 2 (D2) and the ADORA2A system are more sensitive in female than in male individuals [32,33]. Dopamine signaling has been suggested as a therapeutic target of dyslipidemia, showing cardioprotective effects [32]. Caffeine treatment has been shown to increase the expression of D2 protein in female but not in male individuals [33]. Based on our data, we suggest that not only do D2 but also the ADORAs modulate the environmental response of the sex-specific physiological mechanism.

We found a novel gene-environment interaction of the *ADORA* genetic variants and coffee intake on dyslipidemia in a Korean population. However, further, larger studies are warranted to replicate the findings. In addition, while our study did not consider how subjects consumed coffee and how much caffeine was present owing to the limited information in the original cohort, we appreciate the importance of further studies including those parameters. Although it has been reported that the addition of milk, the type of coffee bean, and the type of roasting method do not alter antioxidant activity [34], it may be important to consider these factors to perform an in-depth analysis. Lastly, our analysis did not consider physical activity as a confounding factor, although it has been shown to influence blood lipid profiles [35,36]. Additional confounding factors, such as physical activity, may need to be considered for further analysis.

#### **5. Conclusions**

This study demonstrated that a subset of genetic variants in the *ADORA* gene family influences the association between coffee intake and dyslipidemia risk in a sex-specific manner. As a first study to elucidate the effect of coffee intake on dyslipidemia risk in terms of genetic variability in the *ADORA* gene family, important avenues of detailed research are available. This includes deep understanding of the functional mechanisms on the genetic variants in the *ADORA* gene family in response to coffee intake, potentially aiding prevention and management of dyslipidemia among individuals vulnerable to the disease.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/12/2/493/s1, Table S1. Association between genetic variants in ADORA gene family and the risk of dyslipidemia.

**Author Contributions:** Conceptualization and investigation: J.H. and Y.J.P.; data curation and formal analysis: J.H., J.S. and J.-Y.H.; writing—original draft preparation: J.H.; writing—review and editing: J.S., Y.J.P., and J.-Y.H.; funding acquisition: Y.J.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by Basic Science Research Programs through the National Research Foundation (NRF) funded by the Korea government (2018R1D1A1B07051274) to Y.J.P., J.H. and J.S. were supported by Brain Korea 21 plus project (22A20130012143).

**Acknowledgments:** Data in this study were from the Korean Genome and Epidemiology Study (KoGES; 4851-302). National Research Institute of Health, Centers for Disease Control and Prevention, Ministry for Health and Welfare, Republic of Korea.

**Conflicts of Interest:** The authors declare no conflict of interest.
