Interaction between CETP polymorphism and dietary insulin index and load in relation to cardiovascular risk factors in diabetic adults

Gene–diet interactions may play an important role in the inter individual diversity observed in on cardiovascular disease (CVD) risk factors. Therefore, in the current study, we examined the interaction of CETP TaqB1 polymorphism with dietary insulin index and load (DII and DIL), in altering on CVD risk factors among type 2 diabetes mellitus (T2DM). In this cross-sectional study, blood samples were collected from 220 type 2 diabetic patients (134 females and 86 male) with a mean age of 52.24 years in Tehran, Iran. DIL and DII were obtained via validated food-frequency questionnaire (FFQ). Taq1B polymorphism was genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Biochemical markers including total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglyceride (TG), superoxide dismutase (SOD), C-reactive protein (CRP), total antioxidant capacity (TAC), pentraxin-3 (PTX3), isoprostaneF2α (PGF2α). interleukin 18 (IL18), leptin and ghrelin were measured by standard protocol. Patients with B1B1 genotype had lower lipid profiles include LDL/HDL (P < 0.001) and TG (P = 0.04) when they consumed diets higher on the DIL and DII index. Moreover, carriers of B2B2 genotype who were in the last tertile of DIL had higher antioxidant and inflammatory markers include SOD (P = 0.01), PGF2α (P = 0.04) and CRP (P = 0.02). Further, a significant interaction between CETP TaqB1 and DII was shown in terms of WC (P = 0.01), where the highest WC were observed in B2B2 genotype carriers following a DII score. However, the highest inflammatory and antioxidant markers include CRP (P = 0.04), TAC (P = 0.01), SOD (P = 0.02), and PGF2α (P = 0.02) were observed in B2B2 genotype carriers when they consumed diets higher on the DII index. Based on the current study, it could be proposed that CETP polymorphism may be associated with CVD risk factors in T2DM patients with high following insulin indices, including DII and DIL. It seems that CETP Taq1B polymorphism can invert the result produced by insulin. This conclusion illustrates that the CETP Taq1B B1 allele could counteract the CVD risk induced by high DII and DIL.


Results
General characteristics and dietary intake according to CETP rs708272 genotype. In the current study, 220 patients with T2DM were evaluated. The genotype distributions were within HWE (P-value > 0.05). Table 1 shows the general characteristics based on each participant's genotype. No significant difference was identified in the age, anthropometric data, physical activity and dietary intake between the three genotype groups.

Association between baseline characteristic and metabolic markers with dietary insulin indices.
A statistical analysis of the basic information of patients, among DIL and DII tertiles, are presented in Tables 2 and 3 respectively. Higher tertiles of DIL and DII was associated with higher DIL and DII score respectively (P = 0.001). Individuals following a DIL (P = 0.001) and DII (P = 0.006) was more likely to be male. Subjects  Tables 4 and 5 show the interactions between CETP TaqB1 polymorphism and DIL and DII on anthropometric indices and several biochemical markers. Interactions between CETP TaqB1 polymorphism and DIL were significant in terms of WC (P 1 -interaction = 0.07, P 2 -interaction = 0.04) after adjusting potential confounders: carriers of B2B2 genotype who were in the last tertile of DIL had higher WC. Besides, CETP TaqB1 polymorphism and DIL interactions were significant in terms of lipid markers include LDL/HDL (P 1 -interaction < 0.001, P 2 -interaction < 0.001), TG (P 1 -interaction = 0.03, P 2 -interaction = 0.04), and HDL (P 1 -interaction = 0.03, P 2 -interaction = 0.01) in both crude and adjustment model: carriers of the B1B1 genotype had lower LDL/HDL, TG, and higher HDL when they consumed diets higher on the DIL index.

Discussion
The key findings of the current study were the significant interaction result of CETP rs708272 polymorphism with DIL and DII on obesity indices (WC and BMI), lipid profiles (TG, HDL, LDL/HDL), inflammatory markers (IL-18, CRP, and PGF2α), and antioxidant markers (TAC and SOD) in T2DM patients.
In the present study, B2B2 genotype carriers in the last tertile of DII and DIL were have greater WC as a central obesity marker. CETP Taq1B polymorphism was found to be able to increase the association between dietary insulin indices and obesity. Although several studies have reported an association between DIL and DII scores and obesity 26,27 , the interaction between DII and DIL with CETP polymorphism on obesity was not evaluated yet. A high score of DIL and DII may lead to greater body fat formation by promoting pre-adipocytes differentiation and proliferation to adipocytes 26,27 . Besides, a higher insulin indices score may cause reductions in insulin sensitivity and lipolysis and body fat accumulation by increasing insulin growth factor-1 (IGF-1) [28][29][30] . Additionally, CETP polymorphisms was associated with an elevated risk of obesity and obesity-related diseases [31][32][33] . Although plasma CETP activity has been reported to be elevated in obese subjects [34][35][36][37][38] , this elevation is eliminated with the   35,39,40 . Experimental studies reported that expression of CETP in a diabetic obese animal that cannot normally express CETP, prevented the development of atherosclerotic lesions when they consumed diet-induced obesity 41 . Although, B2 allele of this polymorphism is associated with decreased CETP activity 42,43 and CETP concentration 44,45 . Moreover, previous studies, that suggested there is an inverse association between plasma glucose and CETP activity in T2DM patients 46,47 . Therefore, in our study diabetic patients carriers, B2 allele with reduced CETP activity who consume a diet with higher insulin index which might be associated with higher plasma glucose may act synergistically to elevate a patient's susceptibility to impaired postprandial metabolism, which relates to obesity 48 . The present results also demonstrated the highest inflammatory factors (CRP and PGF2α) in the B2B2 genotype carriers who follow a DIL score and DII. As indicated in the findings, there was a significant interaction between Taq1B CETP polymorphism and DII/DIL in association with anthropometric indices. Numerous findings have reported obesity as causing chronic low-grade inflammatory disorder, contributing to the progression of T2DM and CVD 49 . In these conditions, human adipose tissue secretes a high level of inflammatory markers, including IL-18, CRP, and PGF2α 50 . A further novel finding is Taq1B polymorphism was able to inverse the association of DII and DIL and oxidant status, so that the highest TAC and SOD was observed in the B2B2 genotype following a DIL and DII. However, there is no available study about the relation between DIL/DII and CETP polymorphism interaction with antioxidant status, several studies have reported that insulin concentrations and insulin resistance lead to an imbalance between oxidant and antioxidant systems, a condition known as oxidative stress 51 . In recent years, oxidative stress has been implicated in T2DM pathogenesis via reduced  52 . It seems that CETP Taq1B polymorphism can invert the result produced by insulin. Finally, another promising finding was the significant interaction between Taq1B polymorphism and DII/DIL on lipid profile markers, including TG, HDL, and LDL/HDL. The lowest TG and LDL/HDL, and the highest HDL, were observed in the B1B1 genotype carriers following a DIL and DII. In line with our study, systematic reviews showed that the B1B1 genotype is associated with a better response to nutritional interventions, compared with carriers of B2 alleles 53 . There has been limited study of interaction between Taq1B polymorphism and dietary intake. In this regard, Gammon et al. has showed a significant interaction, where B1B1 homozygotes had a lower TG/HDL ratio after a kiwifruit intervention, compared to a control diet, while B2 carriers were not affected 54 . Nahid Ramezani-Jolfaie et al. revealed that, for diabetic patients, dietary oil treatments would be more helpful (lower LDL; HDL, TG; HDL, TC; HDL, Insulin, and HOMA-IR) among subjects with B1B1 alleles than among B2 allele carriers 55 . Some authors have also suggested that subjects with B1B1 allele of CETP polymorphism showed a better response in regards to high carbohydrate dietary interventions. Overall these findings are in accordance with findings reported by Juan Dua et al., demonstrating that males with CETP Taq1B B1B1 allele have higher apo A-I and HDL concentrations after following a high carbohydrate and low fat (HC/LF) diet for Table 5. Mean values of CVD risk factors across CETP genotypes (B1B, B1B2, B2B2) based on low and high DII intake. Mean values of cardiovascular risk factors across CETP genotypes (B1B1, B1B2, B2B2) based on tertiles of DII intake. P1: P-value for curd model; P2: P-value for the adjusted model by age, gender, physical activity, smoking, alcohol consumption, and familial history of diabetes. Bold values denote statistical significance at the p < 0.05 level.

Limitation and strength.
Limitations of the present study including the cross-sectional design, so any causality cannot be argued; the use of FFQ for dietary assessing, which may have resulted in memory bias; small sample size, which may have led to weak statistical to determine significant results. Furthermore, our participants were from the Iranian country which may not be generalized due to racial and regional differences (52). Due to financial limitations, it was not possible to perform western blot analysis to determine whether rs708272 SNP alters the expression of CETP. Despite the limitations mentioned above, this is the first effort to study the interaction between CETP rs708272 polymorphism and dietary insulin indices on cardio-metabolic risk factors. Recognition of these gene-diet interactions could be determining in prescribe personalized nutritional recommendations for the improvement and management of CVD risk in T2DM patients. Finally, these results can be used in combination with a patient's genetic history to provide more applicable and tailored nutritional advice for preventing or attenuating cardiovascular disease in T2DM patients.

Conclusion
Based on the current study, it could be proposed that CETP polymorphism may be associated with CVD risk factors in T2DM patients with high following insulin indices, including DII and DIL. This conclusion illustrates that the CETP Taq1B B1 allele could counteract the CVD risk induced by high DII and DIL. This could be critical for clinical diagnosis and gene-based therapy. Due to the limited nature of the study, further research with larger sample size is warranted to assess the results on other populations.

Methods
Study population. A cross-sectional study was designed with 220 diabetic patients in Tehran, Iran, who had participated in a large study conducted previously 59 . Diabetic patients who had fasting blood sugar > 126 mg/dl or were using glucose-lowering medications without a history of inflammatory diseases, CVD, stroke, and cancers were included in the study. Besides, patients who were pregnant, addict, taking anti-inflammatory medications and using insulin, lactating patients, and also their total calorie intake was not in range between 800 and 4200 were excluded. All study subjects gave their written informed consent and the study was conducted based on the Declaration of Helsinki, and Ethics Committee of the Tehran University of Medical Sciences approved the protocol (no. 15060).
General, anthropometric and physical activity assessments. Information such as age, disease history, and medication was collected from each participant by questionnaire. Anthropometric measurements, including height (m) and weight (kg) were evaluated without shoes, and using a digital scale; waist circumference (WC) was evaluated at the narrowest part of the abdomen. Finally, body mass index (BMI) was computed by dividing weight (kg) by height squared (meters). Physical activity was measured by the short-form International Physical Activity Questionnaire (IPAQ) 60 .
Biochemical assessments. After fasting for 12-14 h, all blood samples were obtained and centrifuged for 10 min at 3000 rpm to extract serums, which were then aliquoted into 1 ml tubes and refrigerated at 70 °C until analysis. The samples were tested using an auto-analyzer BT 1500 (Selectra 2; Vital Scientific, Spankeren, Netherlands). The levels of TG and TC in the blood were determined using an enzymatic technique and commercially available kits (Pars Azmoon, Iran). Serum levels of HDL-C and LDL-C were determined by turbidimetry on a Roche Hitachi analyzer (Roche, Germany). The serum levels of leptin and ghrelin were also determined using the ELISA method (Bioassay Technology Co, China and Mediagnost, Germany, respectively). The ELISA method was used to determine the amounts of inflammatory markers in the blood, such as IL-18 and PTX3 (Shanghai Crystal Day Biotech Co., Ltd). The IL-18 ELISA kit had a sensitivity of 28 ng/l, and the intra-assay and interassay coefficients of variation (CV) were less than 10% and 12%, respectively. The PTX3 ELISA kit has a sensitivity of 0.05 ng/ml, and the intra-assay and interassay CVs were less than 10% and 12%, respectively. ELISA was used to assess hs-CRP levels in the blood (Diagnostic Biochem Canada Inc., London, Ontario, Canada). The intra-assay and interassay CVs were both less than 5% and 9.5%, respectively. Spectrophometry was used to determine the serum's total antioxidant capacity (TAC). The colorimetric approach was used to determine the serum enzymatic activity of superoxide dismutase (SOD) (Cayman Chemical Company, USA). ELISA (Shanghai Crystal Day Biot) was used to determine the concentration of 8-isoprostane F2 in the blood. All assessment was conducted at the Nutrition and Genomics Laboratory at TUMS.

Dietary assessment.
A validated 147-item food-frequency questionnaire (FFQ) was used to obtain a common dietary intake for patients 61 . The FFQ contained 147 food items with standard portion sizes commonly used by Iranians, based upon on measurements widely used in the community (e.g. number of slices for bread, glasses for drinks, plates for rice, etc.) 62 . The portion sizes in the FFQ were converted into amounts of foods consumed. An expert interviewer carried out the FFQ and recorded participant responses. All reported consumption frequencies were converted to grams per day using household measures. The daily consumption of nutrients and