The combined effects of genetic variation in the SIRT1 gene and dietary intake of n-3 and n-6 polyunsaturated fatty acids on serum LDL-C and HDL-C levels: a population based study

  • Tomoko Inamori1,

    Affiliated with

    • Toshinao Goda2,

      Affiliated with

      • Nobuhiko Kasezawa3 and

        Affiliated with

        • Kimiko Yamakawa-Kobayashi1Email author

          Affiliated with

          Lipids in Health and Disease201312:4

          DOI: 10.1186/1476-511X-12-4

          Received: 30 September 2012

          Accepted: 5 January 2013

          Published: 11 January 2013

          Abstract

          Background

          Dyslipidemia due to high total cholesterol, LDL-cholesterol, triglycerides, or low HDL-cholesterol is an important risk factor for coronary heart disease (CHD). Both SIRT1 and PUFAs can influence the expression of genes for nuclear receptors and transcription factors related to lipid metabolism such as LXRα, LXRβ, PPARα, SREBP-1c.

          Methods

          A total of 707 Japanese males and 723 females were randomly selected from the participants who visited a medical center for routine medical check-ups. We analyzed the combined effects of the genotype/haplotype of the SIRT1 gene and dietary n-6/n-3 PUFA intake ratio on the determination of serum lipid levels.

          Results

          We found that the SIRT1 gene marked with haplotype 2 was associated with decreased serum LDL-cholesterol and increased HDL-cholesterol levels. In addition, the associations between the SIRT1 haplotype 2 and decreased LDL-C and increased HDL-C levels were only observed in the low n-6/n-3 PUFA intake ratio group, but not in the high n-6/n-3 PUFA intake ratio group.

          Conclusions

          Our findings indicate that the combination of genetic variation in the SIRT1 gene and dietary n-6 and/or n-3 PUFA intake influence the determination of inter-individual variations of serum levels of LDL-C and HDL-C.

          Keywords

          LDL-cholesterol HDL-cholesterol SIRT1 Fatty acids

          Background

          Dyslipidemia due to high total cholesterol, low density lipoprotein (LDL)-cholesterol, triglycerides, or low high density lipoprotein (HDL)-cholesterol is an important risk factor for coronary heart disease (CHD) [1, 2]. Data from family and twin studies suggest that genetic variations account for 40-60% of the inter-individual variations in plasma lipid levels [2, 3]. In addition to rare mutations that cause familial dyslipidemia, common genetic variants are considered to significantly contribute to the heritability of plasma lipid levels. For example, genome-wide association studies (GWAS) have reported a growing number of new loci involved in lipid metabolism [4, 5]. However, loci identified through GWAS may not fully explain the inter-individual variation in plasma lipid levels.

          Sirtuin 1 (SIRT1) belongs to the sirtuin protein family of nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylases conserved in evolution from bacteria to humans [6, 7]. Human have seven sirtuin family members, SIRT1-SIRT7, which exhibit with different cellular locations, enzyme activities, target substrates and tissue-specificity. Of these, SIRT1 has been most extensively studied. SIRT1 is a nuclear protein and promotes chromatin silencing and transcriptional repression through histone deacetylation. In addition, more than a dozen non-histone proteins serve as substrates for SIRT1. SIRT1 controls numerous physiological processes and protects cells against stress. A number of studies have shown that SIRT1 orthologs are important mediators of the extension of life span observed from yeast to mammals following calorie restriction. During energy crises such as calorie restriction, NAD+ level rise, concomitant with SIRT1 activation [6, 7]. Transgenic mice overexpressing SIRT1 have beneficial calorie restriction-like phenotypes, while down-regulation of SIRT1 accelerates the aging phenotype in mice [8].

          Furthermore, SIRT1 also has an important function in lipid and glucose metabolism, due to deacetylation of a number of nuclear receptors and transcription factors related to lipid and glucose metabolism such as peroxisome-proliferator activated receptor (PPAR)α, PPARγ, peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1-α), liver X receptor (LXR)α, LXRβ, forkhead box O (FOXO), AMP-activated protein kinase (AMPK) and sterol response element-binding protein-1c (SREBP-1c) [611]. Thus, SIRT1 is associated with lipid metabolism, and variations of the SIRT1 gene might affect the determination of inter-individual variations of plasma lipid levels.

          Fatty acids are no longer just sources of energy, but also fine modulators of cellular signaling and metabolism [12]. There is growing evidence of health benefits of consuming certain types of fats including n-6 and n-3 polyunsaturated fatty acids (PUFAs)[12, 13], which are essential fatty acids that are not synthesized de novo by mammals. Several studies have shown that dietary intake of n-6 PUFAs, such as linoleic acid found in vegetable oils, may reduce CHD risk by beneficial effects on serum total cholesterol, LDL cholesterol, and insulin sensitivity [14], while n-3 PUFA derived from fish has also been shown to decrease serum triglyceride and increase HDL-cholesterol, which is associated with more efficient reverse cholesterol transport and a reduced risk of CHD [15, 16]. It is also reported that n-3 PUFA have anti-inflammatory effects [16]. However, the cellular mechanisms underling the beneficial effects of n-6 and n-3 PUFA on lipid profile and CHD prevention are not completely understood. Recently, n-6 and n-3 PUFAs were shown to be critical for modulation of expression in several nuclear receptors and transcription factors, including LXRα, LXRβ, PPARα, SREBP-1c, hepatocyte nuclear factor (HNF)-4α, and nuclear factor-κB (NFκB) [17]. The majority of these genes play key roles in lipid metabolism, and their expression is also modulated by SIRT1 [611]. Furthermore, it was reported that calorie restriction and dietary n-3 PUFA intake induce the similar beneficial effects such as anti-inflammation, preventing obesity and increasing expression insulin sensitivity in mice [18]. Recently, it was shown that the anti-inflammatory effect of n-3 PUFAs might be mediated through activation of AMPK/SIRT1 pathways; because of n-3 PUFAs increased expression, phosphorylation and activity of AMPK in macrophages, which further leaded to SIRT1 over-expression [19]. These data indicate the possibility that dietary n-3 PUFAs intake modify the SIRT1 activity in vivo.

          In the present study, we investigated whether the common variations in the SIRT1 gene are potential contributors to inter-individual variations in serum lipid levels. Furthermore, we analyzed the interaction of the common SIRT1 variants and dietary n-3 and n-6 PUFAs intake on determination of serum lipid levels.

          Results

          Table 1 shows demographic and biochemical characteristics, and dietary intake of our subjects. There was a significant difference in these data between male and female. As such, we analyzed the data separately in male and female. We examined the relationships between the genotypes/haplotypes of the three single nucleotide polymorphisms (SNPs) (rs7069102, rs2273773, rs3818292) in the SIRT1 gene and metabolic phenotypes such as levels of fasting serum total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, glucose, hemoglobin A1c (HbA1c), and body mass index (BMI). These three SNPs were in linkage disequilibrium (LD) with each other (|D’| > 0.87), and we constructed a haplotype using these three SNPs. There were three common haplotypes with frequencies of >16%, which accounted for 98% of all chromosomes in our subjects (see Additional file 1: Table S1, and Table S2).
          Table 1

          Characteristics of the study subjects

           

          Male

          Female

          P-value

          n

          707

          723

           

          Age (years)

          53.8 ± 5.2

          53.0 ± 5.1

          0.075

          BMI (kg/m2)

          23.5 ± 2.9

          22.2 ± 3.1

          <0.0001

          Total Cholesterol (mg/dl)

          209.9 ± 31.4

          217.7 ± 31.4

          <0.0001

          LDL-Cholesterol (mg/dl)

          130.4 ± 29.8

          131.9 ± 30.0

          0.38

          HDL-Cholesterol (mg/dl)

          57.0 ± 16.3

          71.5 ± 17.1

          <0.0001

          Triglyceride (mg/dl)

          137.8 ± 102.5

          86.8 ± 45.8

          <0.0001*

          glucose (mg/dl)

          99.5 ± 15.4

          92.7 ± 10.9

          <0.0001*

          HbA1c (%)

          5.3 ± 0.59

          5.1 ± 0.44

          <0.0001

          Smokers (%)

          42.5

          8.4

          <0.0001

          Dietary intakes

             

          Energy (kcal/day)

          2106.5 ± 567.0

          1615.5 ± 414.9

          <0.0001

          Total fat (%energy)

          25.9 ± 6.0

          28.2 ± 5.7

          <0.0001

          Carbohydrate (%energy)

          57.1 ± 7.8

          56.0 ± 7.0

          0.012

          SFA (%energy)

          6.5 ± 1.8

          7.6 ± 2.0

          <0.0001

          PUFA (%energy)

          6.7 ± 1.7

          7.0 ± 1.6

          0.0019

          n-6 PUFA (%energy)

          5.4 ± 1.5

          5.7 ± 1.4

          0.0004

          n-3 PUFA (%energy)

          1.5 ± 0.49

          1.5 ± 0.44

          0.42

          Alcohol (g/day)

          28.7 ± 31.6

          6.5 ± 14.6

          <0.0001

          BMI body mass index, HbA1c hemoglobin A1c, SFA saturated fatty acid, PUFA polyunsaturated fatty acid.

          Data are expressed as mean ± SD or percentage.

          P-values were calculated by t-test or χ2 test.

          The subject numbers whose data for LDL-Cholesterol, HbA1c, and dietary intake available are 1430, 1408, and 1248, respectively.

          *Statistical tests for triglyceride and glucose levels were calculated on log-transformed values.

          In males, significant associations were observed between LDL-cholesterol level and all three SNPs or haplotype 2, and between HDL-cholesterol level and haplotype 2. In females, significant associations were observed between LDL-cholesterol level and haplotype 2, and between HDL-cholesterol levels and SNP (rs3818292) or haplotype 1. The other metabolic traits including serum total cholesterol, glucose, HbA1c levels, and BMI were not associated with the genotype/haplotype of the SIRT1 gene (Table 2).
          Table 2

          Relationships between genotypes/haplotypes of the SIRT1 gene and metabolic phenotypes

           

          Genotype

           

          Total-Cholesterol

           

          LDL-Cholesterol

           

          HDL-Cholesterol

           

          Triglycerides*

           

          Glucose*

           

          HbA1c

           

          BMI**

            

          n

          (mg/dl)

          n

          (mg/dl)

          n

          (mg/dl)

          n

          (mg/dl)

          n

          (mg/dl)

          n

          (%)

          n

          (kg/m2)

          Male

          rs7069102

          CC

          487

          208.9 ± 31.4

          482

          129.4 ± 29.1

          487

          57.5 ± 16.7

          487

          135.2 ± 105.4

          487

          99.7 ± 15.7

          484

          5.27 ± 0.60

          487

          23.5 ± 2.8

          (intron 4)

          CG

          207

          212.7 ± 31.4

          203

          133.5 ± 31.0

          207

          55.5 ± 15.3

          207

          146.0 ± 97.3

          207

          99.2 ± 14.8

          206

          5.29 ± 0.58

          207

          23.4 ± 2.9

           

          GG

          13

          203.8 ± 31.8

          13

          122.7 ± 34.3

          13

          60.6 ± 13.7

          13

          100.8 ± 55.5

          13

          93.5 ± 12.1

          13

          5.33 ± 0.61

          13

          24.1 ± 7.1

           

          P value

           

          0.067

           

          0.010

           

          0.42

           

          0.017

           

          0.31

           

          0.46

           

          0.55

          rs2273773

          TT

          315

          212.4 ± 33.2

          311

          134.4 ± 30.6

          315

          55.3 ± 15.7

          315

          136.4 ± 103.1

          315

          99.2 ± 14.8

          313

          5.29 ± 0.58

          315

          23.4 ± 2.9

          (exon 5)

          TC

          294

          208.8 ± 29.6

          290

          127.1 ± 28.5

          294

          58.1 ± 16.4

          294

          140.2 ± 106.8

          294

          99.8 ± 16.9

          293

          5.25 ± 0.64

          294

          23.5 ± 2.9

           

          CC

          98

          207.8 ± 30.4

          97

          127.7 ± 30.0

          98

          58.9 ± 17.5

          98

          134.7 ± 87.3

          98

          99.5 ± 12.2

          97

          5.31 ± 0.47

          98

          23.7 ± 2.8

           

          P value

           

          0.32

           

          0.024

           

          0.054

           

          0.86

           

          0.71

           

          0.81

           

          0.40

          rs3818292

          AA

          313

          212.4 ± 33.4

          309

          134.3 ± 30.6

          313

          55.4 ± 15.6

          313

          136.1 ± 103.7

          313

          99.4 ± 14.8

          311

          5.29 ± 0.59

          313

          23.4 ± 2.9

          (intron 5)

          AG

          297

          208.0 ± 29.4

          293

          127.3 ± 28.4

          297

          57.9 ± 16.4

          297

          140.6 ± 106.0

          297

          99.7 ± 16.9

          296

          5.26 ± 0.64

          297

          23.5 ± 3.0

           

          GG

          97

          207.8 ± 30.7

          96

          127.7 ± 30.2

          97

          59.1 ± 17.8

          97

          134.4 ± 87.7

          97

          99.0 ± 12.0

          96

          5.31 ± 0.46

          97

          23.7 ± 2.7

           

          P value

           

          0.27

           

          0.024

           

          0.10

           

          0.70

           

          0.75

           

          0.85

           

          0.34

          haplotype 1

          +

          515

          210.6 ± 32.4

          510

          131.6 ± 30.1

          515

          56.6 ± 16.2

          515

          135.7 ± 102.2

          515

          99.9 ± 16.5

          513

          5.28 ± 0.63

          515

          23.5 ± 2.8

          (C-T-A)

          -

          192

          208.1 ± 28.6

          188

          127.2 ± 28.7

          192

          58.0 ±16.6

          192

          143.2 ± 103.5

          192

          98.2 ± 11.6

          190

          5.28 ± 0.48

          192

          23.5 ± 3.2

           

          P value

           

          0.53

           

          0.150

           

          0.24

           

          0.21

           

          0.34

           

          0.59

           

          0.86

          haplotype 2

          +

          384

          208.0 ± 29.7

          379

          127.4 ± 28.8

          384

          58.1 ± 16.7

          384

          139.8 ± 102.8

          384

          99.7 ± 16.0

          382

          5.27 ± 0.60

          384

          23.5 ± 2.7

          (C-C-G)

          -

          323

          212.2 ± 33.2

          319

          134.1 ± 30.5

          323

          55.6 ± 15.7

          323

          135.3 ± 102.3

          323

          99.2 ± 14.7

          321

          5.29 ± 0.58

          323

          23.5 ± 3.2

           

          P value

           

          0.17

           

          0.011

           

          0.049

           

          0.47

           

          0.60

           

          0.68

           

          0.14

          haplotype 3

          +

          213

          212.5 ± 31.6

          209

          133.2 ± 31.3

          213

          55.5 ± 14.9

          231

          144.4 ± 97.1

          213

          99.1 ± 14.8

          212

          5.30 ± 0.59

          213

          22.4 ± 3.2

          (G-T-A)

          -

          494

          208.8 ± 31.3

          489

          129.2 ± 29.1

          494

          57.6 ± 16.8

          494

          134.9 ± 104.8

          494

          99.6 ± 15.6

          491

          5.27 ± 0.59

          494

          23.5 ± 2.8

           

          P value

           

          0.069

           

          0.055

           

          0.31

           

          0.053

           

          0.57

           

          0.37

           

          0.51

          Female

          rs7069102

          CC

          504

          217.7 ± 31.9

          492

          131.7 ± 30.9

          504

          71.6 ± 17.2

          504

          85.7 ± 43.2

          504

          92.9 ± 11.3

          493

          5.14 ± 0.45

          504

          22.2 ± 3.2

          (intron 4)

          CG

          198

          217.9 ± 30.2

          190

          132.0 ± 28.0

          198

          71.8 ± 17.0

          198

          87.8 ± 50.2

          198

          92.0 ± 9.0

          192

          5.16 ± 0.40

          198

          22.1 ± 2.9

           

          GG

          21

          215.0 ± 31.1

          20

          134.4 ± 26.3

          21

          63.6 ± 15.9

          21

          102.8 ± 59.6

          21

          94.3 ± 17.7

          20

          5.11 ± 0.44

          21

          22.7 ± 2.8

           

          P value

           

          0.63

           

          0.82

           

          0.12

           

          0.13

           

          0.75

           

          0.20

           

          0.50

          rs2273773

          TT

          310

          218.6 ± 32.0

          300

          134.0 ±30.8

          310

          70.0 ± 16.8

          310

          89.9 ± 49.3

          310

          93.3 ± 12.9

          301

          5.17 ± 0.50

          310

          22.2 ± 3.0

          (exon 5)

          TC

          320

          215.6 ± 31.0

          315

          129.0 ± 29.7

          320

          72.5 ± 17.2

          320

          83.5 ± 40.8

          320

          91.8 ± 8.9

          316

          5.12 ± 0.37

          320

          22.1 ± 3.1

           

          CC

          93

          221.7 ± 30.4

          87

          135.0 ± 27.3

          93

          72.8 ± 17.7

          93

          87.6 ± 49.4

          93

          93.4 ±10.0

          88

          5.15 ± 0.44

          93

          22.6 ± 3.4

           

          P value

           

          0.23

           

          0.091

           

          0.096

           

          0.25

           

          0.14

           

          0.70

           

          0.66

          rs3818292

          AA

          303

          219.1 ± 31.9

          294

          134.4 ± 30.4

          303

          69.8 ± 16.9

          303

          90.3 ± 49.1

          303

          93.5 ± 13.0

          295

          5.17 ± 0.50

          303

          22.2 ± 3.0

          (intron 5)

          AG

          309

          214.9 ± 30.8

          305

          128.7 ± 30.1

          309

          72.4 ± 17.0

          309

          83.3 ± 41.4

          309

          91.7 ± 9.1

          305

          5.12 ± 0.37

          309

          22.1 ± 3.2

           

          GG

          111

          221.8 ± 30.9

          103

          133.8 ± 27.9

          111

          73.4 ± 17.8

          111

          86.8 ± 47.6

          111

          93.2 ± 9.2

          105

          5.13 ± 0.42

          111

          22.5 ± 3.30

           

          P value

           

          0.11

           

          0.061

           

          0.047

           

          0.17

           

          0.090

           

          0.94

           

          0.83

          haplotype 1

          +

          515

          216.9 ± 31.8

          504

          131.7 ± 31.1

          515

          70.8 ± 16.8

          515

          86.6 ± 44.2

          515

          92.7 ± 11.1

          505

          5.14 ± 0.45

          515

          22.1 ± 3.1

          (C-T-A)

          -

          208

          219.7 ± 30.1

          198

          132.2 ± 26.9

          208

          73.2 ± 17.7

          208

          87.2 ± 49.6

          208

          92.7 ± 10.5

          200

          5.14 ± 0.41

          208

          22.3 ± 3.1

           

          P value

           

          0.47

           

          0.77

           

          0.045

           

          0.65

           

          0.70

           

          0.43

           

          0.88

          haplotype 2

          +

          408

          216.9 ± 31.0

          397

          130.1 ± 29.4

          408

          72.6 ± 17.2

          408

          84.5 ± 43.0

          408

          92.1 ± 9.1

          399

          5.13 ± 0.38

          408

          22.2 ± 3.2

          (C-C-G)

          -

          315

          218.7 ± 31.9

          305

          134.1 ± 30.6

          315

          69.9 ± 16.9

          315

          89.7 ± 49.0

          315

          93.4 ± 12.9

          306

          5.16 ± 0.50

          315

          22.2 ± 3.0

           

          P value

           

          0.27

           

          0.039

           

          0.065

           

          0.16

           

          0.088

           

          0.85

           

          0.95

          haplotype 3

          +

          216

          217.5 ± 30.4

          208

          132.4 ± 28.0

          216

          71.0 ± 17.1

          216

          88.9 ± 51.5

          216

          92.2 ± 10.2

          209

          5.15 ± 0.41

          216

          22.2 ± 2.9

          (G-T-A)

          -

          507

          217.8 ± 31.8

          494

          131.6 ± 30.8

          507

          71.7 ± 17.1

          507

          85.8 ± 43.1

          507

          92.9 ± 11.2

          496

          5.14 ± 0.45

          507

          22.2 ± 3.2

           

          P value

           

          0.96

           

          0.67

           

          0.47

           

          0.22

           

          0.92

           

          0.29

           

          0.72

          Values are shown as mean ± SD.

          P-values were calculated by multiple linear regression analyses incorporating age, BMI, current smoking and alcohol intake as covariates.

          *Statistical tests for glucose and TG levels were calculated on log-transformed values.

          **P-values for BMI were calculated by multiple linear regression analyses incorporating age, current smoking and alcohol intake as covariates.

          Statistically significant P-values (P < 0.05) are indicated by bold.

          The carriers of haplotype 2 had lower serum LDL-C and higher HDL-C levels than for the non-carriers of haplotype 2, although in females the relationship between HDL-C levels and haplotype 2 was not statistically significant (P = 0.065). These data indicate that haplotype 2 is a beneficial haplotype associated with decreased LDL-cholesterol and increased HDL-cholesterol levels.

          Next, to examine whether dietary n-6 and n-3 PUFA intake modulates the association between SIRT1 haplotypes and LDL-C and/or HDL-C levels, we classified subjects into two subgroups based on the population median of dietary n-6/n-3 PUFA intake ratio (3.79 for males, 3.93 for females). Significant associations between SIRT1 haplotype 2 and LDL-C and/or HDL-C levels were observed in only the group with a low n-6/n-3 PUFA intake ratio, but were not observed in the group with a high n-6/n-3 PUFA intake ratio (Table 3). In the group with a low n-6/n-3 PUFA intake ratio, the association between haplotype 3 and LDL-C levels in male (P = 0.033), and that between haplotype 1 and HDL-C levels in female were also observed (P = 0.022).
          Table 3

          The combined effects of SIRT1 haplotype and n-6/n-3 PUFA intake ratio on serum LDL-C and HDL-C levels

           

          Male

          Female

          Low n6/n3 intake group(<3.79)

          High n6/n3 intake group(≧3.79)

          Low n6/n3 intake group(<3.93)

          High n6/n3 intake group(≧3.93)

          n

          (mg/dl)

          n

          (mg/dl)

          n

          (mg/dl)

          n

          (mg/dl)

          LDL-Cholesterol

          Haplotype 1 (+)

          221

          133.5 ± 31.1

          232

          131.1 ± 29.3

          215

          133.6 ± 30.5

          213

          131.2 ± 31.3

          Haplotype 1 (−)

          88

          127.0 ± 29.8

          74

          129.4 ± 28.6

          93

          131.3 ± 26.2

          84

          131.1 ± 28.1

          P-value

           

          0.13

           

          0.68

           

          0.53

           

          0.87

          Haplotype 2 (+)

          179

          128.0 ± 30.5

          149

          129.4 ± 28.6

          187

          129.7 ± 27.6

          163

          130.2 ± 30.6

          Haplotype 2 (−)

          130

          136.6 ± 30.7

          157

          131.9 ± 29.7

          121

          137.9 ± 31.0

          134

          132.4 ± 30.1

          P-value

           

          0.0089

           

          0.28

           

          0.0085

           

          0.68

          Haplotype 3 (+)

          82

          136.8 ± 30.2

          95

          131.5 ± 31.6

          82

          131.1 ± 25.1

          98

          134.0 ± 30.2

          Haplotype 3 (−)

          227

          129.8 ± 30.9

          211

          130.3 ± 28.1

          226

          133.6 ± 30.6

          199

          129.8 ± 30.5

          P-value

           

          0.033

           

          0.56

           

          0.99

           

          0.49

          HDL-Cholesterol

          Haplotype 1 (+)

          223

          55.7 ± 16.8

          234

          56.8 ± 15.9

          218

          72.1 ± 17.2

          220

          69.0 ± 16.7

          Haplotype 1 (−)

          89

          58.3 ± 16.7

          77

          57.0 ± 16.0

          96

          76.9 ± 17.8

          91

          69.5 ± 17.4

          P-value

           

          0.16

           

          0.72

           

          0.022

           

          0.92

          Haplotype 2 (+)

          181

          57.6 ± 17.1

          152

          57.4 ± 16.4

          190

          75.5 ± 17.4

          170

          69.4 ± 17.1

          Haplotype 2 (−)

          131

          54.8 ± 16.4

          159

          56.4 ± 15.4

          124

          70.5 ± 17.4

          141

          68.9 ± 16.7

          P-value

           

          0.079

           

          0.27

           

          0.032

           

          0.83

          Haplotype 3 (+)

          84

          53.8 ± 14.9

          97

          56.6 ± 15.0

          84

          71.3 ± 17.1

          104

          69.6 ± 17.7

          Haplotype 3 (−)

          228

          57.4 ± 17.4

          214

          57.0 ± 16.3

          230

          74.4 ± 17.6

          207

          68.9 ± 16.5

          P-value

           

          0.14

           

          0.90

           

          0.15

           

          0.72

          Values are shown as mean ± SD.

          P-values were calculated by multiple linear regression analyses incorporating age, BMI, current smoking and alcohol intake as covariates.

          Statistically significant P-values (P < 0.05) are indicated by bold.

          These findings indicate that the combination of genetic variations in the SIRT1 gene and dietary n-6 and/or n-3 PUFA intake influence the determination of inter-individual variations of serum levels of LDL-C and HDL-C.

          Discussion

          Plasma levels of high LDL-C and low HDL-C are considered major determinants of susceptibility to CHD in the general population. The determination of plasma lipid levels is controlled by multiple pathways and influenced by complex interactions between many different genes and environmental factors such as diet intake.

          In the present study, we found that the combination between the SIRT1 gene marked with haplotype 2 and low n-6/n-3 PUFA intake ratio was related to beneficial effects on serum lipid profile, a decreased LDL-C and an increased HDL-C levels, in both males and females. Both SIRT1 and PUFAs can influence the expression of genes for nuclear receptors and transcription factors related to lipid metabolism including LXRα, LXRβ, PPARα, SREBP-1c [911, 17]. Recently, it was reported that n-3 PUFAs activate AMPK/SIRT1 pathways in macrophages [19]. These data indicate the possibility that the activity or function of SIRT1 might be modified by dietary n-3 PUFAs intake. In addition, it remains possible that the activity of SIRT1 marked with haplotype 2 is more easily increased by n-3 PUFA intake without haplotype 2. At present, we have no direct evidence that the activity of SIRT1 was influenced by dietary n-3 PUFA intake, and the SIRT1 gene marked with haplotype 2 in this study caused alterations in the activity and/or function of SIRT1 deacetylase.

          Genetic variants of the SIRT1 gene have been shown to be associated with human diabetes and obesity-related phenotypes in several previous studies [2022], while only a few genetic association studies for the SIRT1 gene and lipid metabolism have been reported [23]. Almost all genetic variants detected in the SIRT1 gene were synonymous, only a few variants with possible functional changes were reported in the promoter region [24]. The three SNPs that we analyzed in this study are also non-functional variants. And we could not detect associations between genotype/haplotype of the SIRT1 gene and diabetes and obesity-related phenotypes including serum glucose levels, HbA1c, and BMI in this study (Table 2). However, it remains possible that the SIRT1 gene marked with haplotype 2 might exist in linkage disequilibrium with other new functional variants.

          Further in vivo and in vitro studies are needed to assess the role for dietary n-3 or n-6 PUFA intake and genetic variation marked with haplotype 2 in the SIRT1 gene on changes in SIRT1 expression or function.

          The lower saturated fat (meat) and higher n-3-PUFA (fish) in the Japanese diet was suggested to contribute to the lower prevalence of hypercholesterolemia and lower risk of CHD [25]. Furthermore, the beneficial SIRT1 variant marked with haplotype 2 is rather common in the Japanese population (the frequency of haplotype 2 is 0.33). Future studies are required to ascertain whether the combination between the SIRT1 gene marked with haplotype 2 and low n-6/n-3 PUFA intake ratio can produce beneficial effects on serum lipid profile in other populations containing young people, children, or other people with different dietary habits. Prospective cohort studies are also required to determine the interactions between genetic variations of the SIRT1 gene and dietary PUFA on serum lipid profile. It is important to determine the potential for interactions between genetic and modifiable environmental factors such as dietary nutrient intake to establish a preventive method for common diseases such as dyslipidemia and CHD.

          Conclusion

          We found that the genotype/haplotype of the SIRT1 gene is associated with serum LDL-C and HDL-C levels, and that the variant marked with haplotype 2 is associated with decreased LDL-cholesterol and increased HDL-cholesterol levels. In addition, the associations between the SIRT1 haplotype 2 and decreased LDL-C and/or increased HDL-C levels were only observed in the low n-6/n-3 PUFA intake ratio group, but not in the high n-6/n-3 PUFA intake ratio group. These findings indicate that the combination of genetic variations in the SIRT1 gene and dietary n-6 and/or n-3 PUFA intake can influence the determination of inter-individual variations of serum levels of LDL-C and HDL-C. An understanding of the interactions of genetic and environmental factors on the prevalence of dyslipidemia is useful for prediction and prevention of CHD, as high LDL-C and low HDL-C levels are important risk factors for CHD.

          Subjects and methods

          Subjects

          A total of 707 Japanese males and 723 females were randomly selected from participants who visited a medical center near the University of Shizuoka for routine medical check-ups. Subjects were all Japanese, and ranged from 45–65 years (mean age, 53.8 ± 5.2 males, 53.0 ± 5.1 females). People taking medication for dyslipidemia and/or diabetes were excluded from the study subjects. After overnight fasting, blood samples were collected from each subject. Written informed consent was obtained from all subjects and this study was approved by the Ethics Committee of the University of Shizuoka.

          DNA analysis

          Genomic DNA was isolated from peripheral leucocytes by the phenol extraction method. We analyzed the genotypes and haplotypes of three tag SNPs (rs7069102 [intron 4], rs2273773 [exon 5, Leu27Leu], rs3818292 [intron 5]) in the SIRT1 gene, which were selected from the HapMap database (http://​hapmap.​ncbi.​nlm.​nih.​gov/​index.​html.​en), based on their minor allele frequencies (MAF) in the Japanese population and previous reports describing the SIRT1 polymorphism [20, 23]. The SIRT1 gene is localized to chromosome region 10q21.3 with 9 exons. To date, approximately 100 common SNPs (MAF≧ 0.05) have been on the dbSNP database (http://​www.​ncbi.​nlm.​nih.​gov/​snp?​term). The majority of SNPs exist in intron or untranslated regions.

          The genotypes of these three SNPs were determined for each subject using the PCR-restriction fragment length polymorphism method. The haplotypes and their frequencies were estimated by the maximum-likelihood method with an expectation-maximization-based algorithm using the SNPAlyze program (Dynacom, Tokyo, Japan).

          Dietary assessment

          Dietary intake was assessed using a brief-type self-administered diet history questionnaire (BDHQ). The BDHQ was developed based on the self-administered diet history questionnaire (DHQ), which had been validated using three different standard methods for dietary assessment [26, 27]. The BDHQ were designed to obtain dietary habits for the previous month from validating dietary intake for 58 food and beverage items which are commonly consumed in general Japan populations [26, 27]. With respect to n-3 and n-6 PUFA, the majority food sources are α-linolenic acid (vegetable oil), EPA (fish and shellfish), DHA (fish and shellfish) and linoleic acid (vegetable oil), arachidonic acid (fish, organ meats and egg), respectively. Intake of fat, carbohydrate, protein, and fatty acids was expressed as percentages of the total non-alcohol energy intake. Dietary data were available for 1248 subjects (87.3%) in this study.

          Statistical analyses

          The relationships between genotypes/haplotypes of the three SNPs of the SIRT1 gene and metabolic parameters including serum lipid and glucose levels were analyzed by multiple linear regression analyses incorporating age, BMI, alcohol intake, and smoking status as covariates. Statistical analyses were performed using the JMP 9 software package (SAS Institute, Cary, NC, USA). The coefficients of linkage disequilibrium (LD) value (|D’| and r2) among three SNPs were calculated by using the SNPAlyze program (Dynacom, Tokyo, Japan).

          Abbreviations

          LDL: 

          Low density lipoprotein

          HDL: 

          High density lipoprotein

          CHD: 

          Coronary heart disease

          GWAS: 

          Genome-wide association study

          SIRT1: 

          Sirtuin 1

          PPAR: 

          Peroxisome-proliferator activated receptor

          PGC-1α: 

          Peroxisome proliferator-activated receptor gamma coactivator 1-alpha, LXR, Liver X receptor

          FOXO: 

          Forkhead box O

          AMPK: 

          AMP-activated protein kinase

          SREBP: 

          Sterol response element-binding protein

          PUFA: 

          Polyunsaturated fatty acid

          SNP: 

          Single nucleotide polymorphism

          HbA1c: 

          Hemoglobin A1c

          BMI: 

          Body mass index

          LD: 

          Linkage disequilibrium.

          Declarations

          Acknowledgments

          We are grateful to the subjects for their participation in this study. We thank Prof. Satoshi Sasaki (The University of Tokyo) for his advices on dietary assessment. This study was supported by the Global COE program from the Ministry of Education, Culture, Sport, Science and Technology of Japan (MEXT).

          Authors’ Affiliations

          (1)
          Laboratory of Human Genetics, School of Food and Nutritional Sciences, Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka
          (2)
          Laboratory of Nutritional Physiology, School of Food and Nutritional Sciences, Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka
          (3)
          Department of Data Managements for Health Evaluation & Promotion, Shizuoka Medical Center

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          Copyright

          © Inamori et al.; licensee BioMed Central Ltd. 2013

          This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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