Interaction between dietary total antioxidant capacity and BDNF Val66Met polymorphism on lipid profiles and atherogenic indices among diabetic patients

Brain-derived neurotrophic factor (BDNF) belongs to the “neurotrophin” family of growth factors, and it has recently been associated to cardiovascular disease (CVD). We anticipated that BDNF Val66Met polymorphisms may alter CVD risk markers such as serum lipid profile differences, and interaction with total antioxidant capacity of diet (DTAC) could alter these clinical parameters. This cross-sectional study consisted of 667 diabetic patients (39.7% male and 60.3% female). DTAC was calculated by international databases. 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. Atherogenic indices (AIP, AC, CR-I, CR-II) were calculated. Genotyping of the BDNF Val66Met polymorphisms was conducted by the real-time PCR–RFLP method. The gene-diet interactions were evaluated using a generalized linear mode (GLMs). Carriers of the Val/Met genotype who were in the higher median intake of FRAP had lower HDL (P:0.04) and higher TG (P:0.005), AIP (P:0.02) and AC (P:0.02) index compared to Val/Val genotypes with lower median intake. Moreover, diabetic patients with Val/Met genotype who consumed higher ORAC intake had increased odds for anthropometric indices (BMI (P:0.01) and WC (P:0.03)), lipid profiles (TG) (P:0.01), and atherogenic index (AIP) (P:0.02), also decreased odds for HDL (P:0.03) concentration compared to reference group whit lower ORAC intake. Individuals with Val/Met genotype who consumed higher TRAP intake had increased odds for WC (P:0.04), TC (P:0.001), TG (P < 0.001), AIP (P < 0.001) and AC (P < 0.001). Finally, Val/Met patients with a higher median intake of TEAC had higher TG (P:0.02), AIP (P:0.009) and AC (P:0.03) compared to the reference group whit lower TEAC intake. Our study showed that Val/Met genotype had also the highest lipid profile and atherogenic indices even in the highest adherence to DTAC. While it seems that the presence of the Val/Val wild-type and BDNF Met/Met homozygotes in diabetic patients with a high DTAC is a protective factor.

Association between population characteristics, biochemical parameters between DTAC and BDNF Val66Met polymorphism. We found that there was no significant association between lipid profiles and atherogenic indices among BDNF Val66Met genotypes (p > 0.05) ( Table 1).
Lipid profiles and atherogenic indices among DTAC groups (ferric reducing-antioxidant power (FRAP), total radical-trapping antioxidant parameter (TRAP), total reactive antioxidant potential (TEAC), and oxygen radical absorbance capacity (ORAC)), is presented in Tables 2 and 3. An individual with higher adherence to FRAP (P = 0.02), TEAC (P = 0.04), and ORAC (P = 0.01) had lower TAC concentrations. Moreover, patients with a higher intake of TRAP (P = 0.03) were more likely to have higher IL-18 concentrations.

Discussion
The key findings of the current study were the significant interaction result of BDNF Val66Met polymorphism with DTAC on lipid profile and atherogenic indices in T2DM patients. High DTAC intake modified the association of the BDNF Val66Met genotypes with the odds of higher lipid profile and atherogenic indices. Particularly, we revealed that increased DTAC did not influence the negative consequences of the Val/Met genotype. While it seems that the presence of the Val/Val wildtype and BDNF Met/Met homozygotes in diabetic patients with a high DTAC is a protective factor.
In this present study, we revealed that an individual with higher adherence to DTAC had lower TAC concentrations. Moreover, patients with a higher intake of TRAP were more likely to have higher IL-18 concentrations. There have been few investigations on the relationship between DTAC and metabolic indicators such as Table 1. The association between BDNF Val/Met polymorphism with lipid profiles and atherogenic indices in T2DM patients. Data are presented as mean ± standard deviation (SD). BMI body mass index, HDL-c high density lipoprotein cholesterol, LDL-c low density lipoprotein cholesterol, TG triglyceride, CRP C-reactive protein, PTX3 pentraxin-3, IL18 interleukin 18, TAC total antioxidant capacity, SOD superoxide dismutase, PGF2α prostaglandinF2α, FRAP ferric reducing ability of plasma, TRAP total reactive antioxidant potential, TEAC trolox equivalent antioxidant capacity, ORAC oxygen radical absorbance capacity, AIP log (TG/HDL), AC (TC-HDL)/HDL, CRI.II (LDL/HDL), CRI-I (TC/HDL). www.nature.com/scientificreports/ lipid profiles and inflammatory markers; the findings of numerous research have contradicted the findings of the current study 20,[31][32][33][34][35] . In line with our findings, Mozaffari et al. 36 , have shown subjects in the highest tertile of dietary TRAP had higher BMI than those in the lowest tertile. Numerous findings have reported obesity has been linked to a persistent low-grade inflammatory disease, which has been linked to the development of T2DM and CVD 37,38 . In these situations, human adipose tissue secretes a high amount of inflammatory markers, including IL-18, in these circumstances 39 .
We did not find significant association between BDNF rs6267 groups and biochemical markers. BDNF play important role in etiology of diabetes and obesity by probably biological mechanism including controlling food behaviour, energy homeostasis and anorexigenic effects 40 . Some experimental studies have also suggested that   www.nature.com/scientificreports/ hyperphagia, hyperinsulinemia, and higher levels of serum leptin and body weight following a decrease in BDNF levels, among BDNF-knockout mice 41 . In terms of human study, association between Val66Met polymorphism with obesity, dyslipidaemia and diabetes are controversial. For instance, several studies have shown that Met/Met genotype have higher risk for insulin resistance, obesity and dyslipidaemia 24,42,43 . Bonaccorso et al. revealed, Metallele of BDNF Val66Met polymorphism was to be positively associated with serum levels TG and TG/HDL-C ratio 24 . However, some authors have suggested the Met-allele carriers have lower risk for obesity, postprandial glucose and HbA1c levels 25,44,45 . Additionally, also some studies have observed no association with anthropometric indices and lipid profile [46][47][48] . The inconsistent results have been revealed on the association between the Val66Met polymorphism and obesity and lipid profile, proposing that environmental factors like dietary intake may be modify this association. www.nature.com/scientificreports/ In this study, we discovered that a diet high in overall antioxidant capacity is more beneficial in homozygotes carriers than in Val/Met carriers. This is the first research to investigate at how dietary antioxidants interact with the BDNF polymorphism. In addition, only a few studies have examined the relationship between BDNF rs6267 and dietary patterns, as well as food and nutrient intakes 29,30 . According to a pervious study, individuals with BDNF Val/Met and Met/Met had a lower risk for T2DM in low energy intake and especially BDNF Val/Met had a negative association with low-protein, high-carbohydrate, and low-fat diet. In comparison to BDNF Val/Val, BDNF Val/Met reduced the risk of HOMA-IR in low-energy intake but raised the risk of HOMA-B in high-energy intake. HOMA-B is an insulin secretion capacity index that could aim to decrease the incidence of T2DM. When high-energy consumption is combined with BDNF Val/Met, insulin secretion is increased in terms of maintaining normoglycemia condition. As a result, individuals with BDNF Val/Met may also have a higher potential to compensate for the development of T2DM 29 . In this term, another study has shown, total food intake, total caloric intake, and protein intake were not related to the BDNF rs6265 variation. Regarding obesity indicators, although, this variation interacted with PUFA and total food intake. Met allele carriers in men exhibited a higher BMI as their PUFA intake increased, and a smaller waist as their n-3: n-6 PUFA ratio increased. In contrast to heterozygotes, Val/Val homozygous men showed the opposite trend in BMI: BMI dropped with increased PUFA intake and higher n-3:n-6 PUFA ratio increased waist circumference 30 . Furthermore, another study indicated that when Met allele carriers were exposed to a high-CHO diet, their chance of developing carbohydrate-induced hypertriglyceridemia enhanced 49 . In contrast, another study has shown, Val66Met polymorphism did not appear to affect the link between food quality and BDNF serum in terms of depression prediction 50 . Previous research has revealed that dietary intakes alter the relationship between BDNF genotype and obesity-related behaviors, which is corroborated by findings in rats [51][52][53] . As a result, food consumption may influence the relationship between BDNF polymorphism and cardiovascular disease indicators via BDNF expression and serum protein modulation.
On the effect of diet on the BDNF serum, some nutrigenomic studies have done based on diet or other macronutrient induced obesity and healthy diet. For example, a previous study found that high glucose concentrations decreased BDNF release 54 . As a result, blood BDNF levels in T2DM patients were shown to be considerably higher than in healthy controls in humans and were found to be strongly associated with triglyceride levels 55 . In diabetic mice, subcutaneous injections of BDNF were found to considerably improve lipid and glucose profiles 56 . In this regard, experimental studies have revealed that diet-induced obese mice include high-fat and high-sugar diets 53,57,58 or n-3 PUFA deficient diets 51 and chronic high-fat DIO mice 53 lowered BDNF expression in the hippocampus by more than 30%, which has been associated to weaker inhibitory regulation of food consumption and, as a result, promoted obesity-related phenotype, while low-fat mice showed no difference.
We observed that the presence of the Val/Val wild-type and BDNF Met/Met homozygotes in diabetic patients with a high DTAC is a protective factor. Antioxidant-rich diets may affect previously unknown biological processes, altering vulnerability to cardiovascular disease. Antioxidant-rich diets have been demonstrated to have a positive influence on metabolic syndrome components, cardiovascular disease risk factors, and obesity-related aspects in several epidemiological investigations 36,59,60 . Reduced inflammation and oxidative stress, increased leptin gene expression, appetite regulation, adipocyte metabolism regulation, and suppression of nuclear factor-B factor are all probable mechanisms 36,61,62 .
We investigated the nutrigenomic research on BDNF serum because there was no nutrigenetics study in this term. In adult rats, fish oil treatment resulted in a considerable increase in BDNF expression 52 . Furthermore, administration of whole-grain (WG) rye has been demonstrated to upregulate BDNF levels. When compared to white wheat flour-based bread meals raised BDNF levels by 27% after fasting 63 . Furthermore, prebiotic feeding elevated the expression of BDNF and peptide YY 64 . Furthermore, there was a 'very probable' rise in BDNF levels with protein supplementation 65 . A Mediterranean-style diet 66 , omega-3 fatty acids 67,68 , and even vitamin E and refined flavonoids consumption have all been associated with increased concentrations of brain BDNF. There are some possible mechanisms for these favourable effects in our study and nutrigenomic studies include similar  www.nature.com/scientificreports/ components. Endothelial dysfunction is prevented by a high antioxidant diet, which is linked to reduced levels of pro-inflammatory cytokines in the plasma [69][70][71][72] Moreover, pro-inflammatory cytokines like IL-6 and TNFmay also suppress BDNF expression 73 . Compliance to these interventions would be predicted to be related with greater plasma BDNF concentrations under this mechanism. These antioxidant components increase BDNF levels and phosphorylation of the CREB pathway [74][75][76] . Although we observed being in BDNF Val/Val wild-type and BDNF Met/Met homozygotes reduced lipid markers and atherogenic indices between homozygotes participant, heterozygotes have shown increased these factors even in high DTAC. These results go beyond previous reports, showing the negative effect of heterozygotes BDNF mic opposite of homozygotes. The heterozygotes BDNF val-66met genotype is correlated with cortical morphology that differs from that of BDNF val66met homozygotes. The BDNF Val/Met genotype, in particular, may affect brain tissue volumes and neurodevelopment, resulting in phenotypic differences between BDNF Val/Val wildtype and BDNF Met/Met homozygotes [77][78][79] . Interestingly, 5-HT turnover was impaired in heterozygous BDNF+/− mice with lowered BDNF expression and resulting in increased food intake and obese phenotypes 80 . Kernie et al. found that heterozygous BDNF/-mice with low BDNF mRNA expression gain 300% more body fat and develop obesity than homozygous BDNF/-mice with high BDNF mRNA expression 81 . Consequently, diet-induced dyslipidemia may be exacerbated by the downregulation of BDNF in heterozygotes individuals. Besides, these discrepancies may be attributed to variations in food choices, fortification, and preferences, which could also impact antioxidant intake through various dietary sources, as well as some changes in antioxidant activity and availability that occur during food processing and preparation [82][83][84][85] . The unfavorable impact found in our study between Val/Met genotypes could be related to a higher intake of high-calorie antioxidant-rich foods and a higher energy intake, since we discovered in our study that greater DTAC consumption was associated with higher carbohydrate intake in diabetic patients 86 . We did not separate different sources of TAC, also based on previous studies source of TAC may have significant effect on biochemical markers 87 .

Limitations and strengths
limitations of the present study including the cross-sectional design, so any causality cannot be argued; the use of FFQ for dietary assessing. Due to financial limitations, it was not possible to perform a western blot analysis to determine whether rs6265 SNP alters the expression of BDNF. markers. Furthermore, our participants were from the Iranian country which may not be generalized due to racial and regional differences. Despite the limitations mentioned above, this is the first effort to study the interaction between BDNF Val66Met polymorphism and DTAC on lipid profiles and atherogenic indices. 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
However, this study has several strengths among which it should be emphasized that is the first study of the geneenvironment interaction in diabetic patients exploring how BDNF polymorphism (Val66Met) affects the diet in correlation with lipid profiles and atherogenic indices, adding important information to previous studies that assessed dietary habits and lipid markers in diabetic patient's groups without considering genetic implications. Further functional studies are necessary to confirm the exact mechanism through which this SNP influences food intake regulation. www.nature.com/scientificreports/

Method
Study population. The current cross-sectional study was carried out on 667 T2DM patients who were referred from diabetes referral clinics in Tehran, Iran. Our study comprised diabetic patients with fasting blood sugar levels of > 126 mg/dl or who were on glucose-lowering medicines. The complete inclusion and exclusion criteria, demographic, physical activity (METs) information, and anthropometric measurements (body mass index (BMI) and WC), were taken based on our previous larger investigation 88 . The International Physical Activity Questionnaire (IPAQ) short form was used to assess physical activity. The reliability and validity of the IPAQ has previously been evaluated in Iranian adolescents 89 . All of the patients were asked to give their informed permission. 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).
Biochemical assessments. The study participants' venous blood samples were taken after they had fasted for 12 h. The levels of HDL-C and LDL-C in the blood were measured using a Roche Hitachi analyzer using turbidimetry (Roche, Germany). The ELISA approach was also used to determine the serum levels of leptin and ghrelin (Bioassay Technology Co, China and Mediagnost, Germany, respectively). The number of inflammatory markers in the blood, such as IL-18 and PTX3, was measured using the ELISA method (Shanghai Crystal Day Biotech Co., Ltd). The intra-assay and interassay coefficients of variation (CV) were less than 10% and 12%, respectively, for the IL-18 ELISA kit, which had a sensitivity of 28 ng/l. The intra-assay and interassay CVs were less than 10% and 12%, respectively, for the PTX3 ELISA kit, which has a sensitivity of 0.05 ng/ml. The levels of hs-CRP in the blood were measured using an ELISA kit (Diagnostic Biochem Canada Inc., London, Ontario, Canada). The intra-assay and interassay CVs were both less than 5% and 9.5%, respectively. Both the intra-and inter-assay CVs were less than 5% and 9.5%, respectively. The total antioxidant capacity of the serum was determined using specttrophometry (TAC). The serum enzymatic activity of SOD was measured using a colorimetric method (Cayman Chemical Company, USA). The concentration of 8-isoprostane F2 in the blood was measured using an ELISA (Shanghai Crystal Day Biot). The Nutrition and Genomics Laboratory at TUMS was used to conduct all of the tests.

Atherogenic indices of plasma (AIP) and lipid ratio assessment. The atherogenic indices of plasma
were calculated using the logarithmic ratio of (TG to HDL-C) (AIP). Furthermore, Olamoyegun et al. invented the lipid ratio, which is calculated using the following formula: CRI-I = TC/HDL-C, CRI-II = LDL-C/HDL-C, AC = (TC − HDL-C)/HDL-C.
Dietary assessment and DTAC calculation. Dietary data were analyzed using a standard semi-quantitative FFQ that included 147 food categories and was specifically prepared for usage in Iran 90 . Face-to-face personal interviews with professional dietitians were used to complete the FFQ. Participants were asked to rate the frequency with which they consumed each food item throughout the previous year. Using household measures, the portion sizes of ingested food products were translated to grams per day.   Low High Figure 2. Interaction between the BDNF Val66Met polymorphism and DTAC (ORAC) intake with regard to TG, HDL, AIP and AC according to the median DTAC, the participants were dichotomized into low and high categories. P 1 = P value with unadjusted (crude) model, P 2 = P value with adjustments for potential confounding factors including (Age, physical activity, sex, smoking, alcohol, energy intake, lipid, and glucoselowering medicines, and family history of diabetes). www.nature.com/scientificreports/ the χ 2 test. Based on their FRAP, TRAP, TEAC, and ORAC scores, the subjects were separated into two groups: low and high intakes. Qualitative variables were compared with one-way ANOVA and analysis of covariance (ANCOVA) in crude and adjusted models respectively. Potential interactions between the rs6265 genotype and DTAC on lipid profiles and atherogenic indices were investigated using the generalized linear models (GLMs) model. Age, physical activity, sex, smoking, alcohol, energy intake, lipid, and glucose-lowering medicines, and family history of diabetes were all used as cofounder factors in adjusted analyses. All stages of our research's analysis were conducted using SPSS software (SPSS Inc., Chicago, IL, USA, version 25). A p-value of less than 0.05 was also considered significant.   Figure 3. Interaction between the BDNF Val66Met polymorphism and DTAC (TEAC) intake with regard to TG, HDL, AIP and AC according to the median DTAC, the participants were dichotomized into low and high categories. P 1 = P value with unadjusted (crude) model, P 2 = P value with adjustments for potential confounding factors including (Age, physical activity, sex, smoking, alcohol, energy intake, lipid, and glucoselowering medicines, and family history of diabetes).  . Interaction between the BDNF Val66Met polymorphism and DTAC (TRAP) intake with regard to TG, HDL, AIP and AC according to the median DTAC, the participants were dichotomized into low and high categories. P 1 = P value with unadjusted (crude) model, P 2 = P value with adjustments for potential confounding factors including (Age, physical activity, sex, smoking, alcohol, energy intake, lipid, and glucoselowering medicines, and family history of diabetes). www.nature.com/scientificreports/