Conjugated Linoleic Acid Isomers Exert Differential Effects on an Adipocyte Model of HIV-associated Lipodystrophy.

BACKGROUND: HIV-associated lipodystrophy is associated with decreased expression of PPAR-γ in adipose tissue. Conjugated linoleic acid (CLA) isomers (cis9, trans11 and trans10, cis12) are putative PPAR-γ agonists, but have not previously been investigated in the context of HIVassociated lipodystrophy. METHOD: 3T3-L1 pre-adipocytes were differentiated in the presence of ritonavir (20 µM as per previous experimental models) and 100 µM cis9,trans11, trans10,cis12 or vehicle control, DMSO. Microarray analysis, RT-PCR, DNA binding ELISA and Oil Red O staining were used to investigate adipocyte gene expression and binding, protein secretion and triglyceride storage. RESULTS: trans10, cis12 + ritonavir altered the expression of 2160 gene transcripts greater than 1.5-fold compared with control, while 257 gene transcripts were altered by cis9,trans11 + ritonavir (P<0.001). trans10,cis12 + ritonavir down-regulated Pparg (-1.55) and Adipoq (-2.95), as well as differentiation (Fcor (-4.78-fold), Arl4a (-4.84), Itga6 (-2.45), Id4 (-2.01)) and triglyceride storage genes (Mrap (- 8.25), Scd1 (-4.34), Lipin1 (-2.52)). Changes in Adipoq were confirmed by RT-PCR (P=0.038) and adiponectin secretion by ELISA (P= 0.003). cis9,trans11 + ritonavir increased PPAR-γ nuclear binding to its gene response element (P=0.038). Both isomers increased triglyceride storage in the presence of ritonavir (P<0.001). CONCLUSION: In the presence of ritonavir, trans10, cis12 appears to be detrimental, while cis9, trans11 was beneficial and may mediate its effects via PPAR-γ. Further research is required to determine the potential role of CLA isomers as therapeutic agents in the management of HIV-associated lipodystrophy.

Microarray Analysis Reveals Altered Lipid and Glucose Metabolism Genes in Differentiated, Ritonavir-Treated 3T3-L1 Adipocytes.

OBJECTIVE: HIV lipodystrophy is characterised by abnormal adipose tissue distribution and metabolism, as a result of altered adipocyte function and gene expression. The protease inhibitor ritonavir is associated with the development of lipodystrophy. Quantifying changes in adipogenic gene expression in the presence of ritonavir may help to identify therapeutic targets for HIV lipodystrophy. METHODS: Affymetrix Mouse Genome 430 2.0 oligonucleotide microarray was used to investigate gene expression in 3T3-L1 adipocytes treated with 20 µmol/l ritonavir or vehicle control (ethanol). Pparg, Adipoq, Retn and Il6 expression were validated by real time RT-PCR. Transcriptional signalling through PPAR-γ was investigated using a DNA-binding ELISA. Changes in adipocyte function were investigated through secreted adiponectin quantification using ELISA and Oil Red O staining for triglyceride storage. RESULTS: Expression of 389 genes was altered by more than 5-fold in the presence of ritonavir (all P < 0.001). Gene ontology analysis revealed down-regulation of genes responsible for adipocyte triglyceride accumulation including complement factor D (Cfd; 238.42-fold), Cidec (73.75-fold) and Pparg (5.63-fold). Glucose transport genes were also down-regulated including Adipoq (24.42-fold) and Glut4 (13.36-fold), while Il6 was up-regulated (10.39-fold). PPAR-γ regulatory genes Cebpa (11.33-fold) and liver-X-receptor α (Nr1h3) were down-regulated. Changes in Pparg, Adipoq and Il6 were confirmed by RT-PCR. PPAR-γ binding to its nuclear consensus site, adiponectin secretion and triglyceride accumulation were all reduced by ritonavir. CONCLUSION: Ritonavir had a significant effect on expression of genes involved in adipocyte differentiation, lipid accumulation and glucose metabolism. Down-regulation of Pparg may be mediated by changes in Cebpa, Lcn2 and Nr1h3.

Genetic predisposition scores for dyslipidaemia influence plasma lipid concentrations at baseline, but not the changes after controlled intake of n-3 polyunsaturated fatty acids.

Inconsistent effects of fish oil supplementation on plasma lipids may be influenced by genetic variation. We investigated 12 single nucleotide polymorphisms (SNPs) associated with dyslipidaemia in genome-wide association studies, in 310 participants randomised to treatment with placebo or 0.45, 0.9 and 1.8 g/day eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) (1.51:1) in a 12-month parallel controlled trial. Effects of risk alleles were assessed as trait-specific genetic predisposition scores (GPS) and singly. GPS were positively associated with baseline concentrations of plasma total cholesterol, low-density-lipoprotein cholesterol and triglyceride (TG) and negatively with high-density-lipoprotein cholesterol. The TG-GPS was associated with 0.210 mmol/L higher TG per risk allele (P < 0.0001), but no effects of single TG SNPs were significant at baseline. After treatment with EPA and DHA, TG-GPS was associated with 0.023 mmol/L lower TG per risk allele (P = 0.72). No interactions between GPS and treatment were significant; however, FADS1 SNP rs174546 C/T interaction with treatment was a significant determinant of plasma TG concentration (P = 0.047, n = 267). Concentration differed between genotype groups after the 1.8 g/day dose (P = 0.026), decreasing by 3.5 (95 % CI -15.1 to 8.2) % in non-carriers of the risk T-allele (n = 30) and by 21.6 (95 % CI -32.1 to -11.2) % in carriers (n = 37), who showed a highly significant difference between treatments (P = 0.007). Carriers of the FADS1 rs174546 risk allele could benefit from a high intake of EPA and DHA in normalising plasma TG.

Interaction between a CSK gene variant and fish oil intake influences blood pressure in healthy adults.

Blood pressure is a heritable determinant of cardiovascular disease (CVD) risk. Recent genome-wide association studies have identified several single-nucleotide polymorphisms (SNPs) associated with blood pressure, including rs1378942 in the c-Src tyrosine kinase (CSK) gene. Fish oil supplementation provides inconsistent protection from CVD, which may reflect genetic variation. We investigated the effect of rs1378942 genotype interaction with fish oil dosage on blood pressure measurements in the MARINA (Modulation of Atherosclerosis Risk by Increasing doses of N-3 fatty Acids) study, a parallel, double-blind, controlled trial in 367 participants randomly assigned to receive treatment with 0.45, 0.9, and 1.8 g/d eicosapentaenoic acid [EPA (20:5n-3)] and docosahexaenoic acid [DHA (22:6n-3)] (1.51:1) or an olive oil placebo for 12 mo. A total of 310 participants were genotyped. There were no significant associations with blood pressure measures at baseline; however, the interaction between genotype and treatment was a significant determinant of systolic blood pressure (SBP) (P = 0.010), diastolic blood pressure (DBP) (P = 0.037), and mean arterial blood pressure (MABP) (P = 0.014). After the 1.8 g/d dose, noncarriers of the rs1378942 variant allele showed significantly lower SBP (P = 0.010), DBP (P = 0.016), and MABP (P = 0.032) at follow-up, adjusted for baseline values, than did carriers. We found no evidence of SNP genotype association with endothelial function (brachial artery diameter and flow-mediated dilatation), arterial stiffness (carotid-femoral pulse wave velocity and digital volume pulse), and resting heart rate. A high intake of EPA and DHA could help protect noncarriers but not carriers of the risk allele. Dietary recommendations to reduce blood pressure in the general population may not necessarily benefit those most at risk. This trial was registered at controlled-trials.com as ISRCTN66664610.

ELOVL2 gene polymorphisms are associated with increases in plasma eicosapentaenoic and docosahexaenoic acid proportions after fish oil supplement.

Fish oil supplementation provides an inconsistent degree of protection from cardiovascular disease (CVD), which may be attributed to genetic variation. Single nucleotide polymorphisms (SNPs) in the elongation-of-very-long-chain-fatty-acids-2 (ELOVL2) gene have been strongly associated with plasma proportions of n-3 long-chain polyunsaturated fatty acids (LC-PUFA). We investigated the effect of genotype interaction with fish oil dosage on plasma n-3 LC-PUFA proportions in a parallel double-blind controlled trial, involving 367 subjects randomised to treatment with 0.45, 0.9 and 1.8 g/day eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (1.51:1) or olive oil placebo for 6 months. We genotyped 310 subjects for ELOVL2 gene SNPs rs3734398, rs2236212 and rs953413. At baseline, carriers of all minor alleles had lower proportions of plasma DHA than non-carriers (P = 0.021-0.030). Interaction between genotype and treatment was a significant determinant of plasma EPA (P < 0.0001) and DHA (P = 0.004-0.032). After the 1.8 g/day dose, carriers of ELOVL2 SNP minor alleles had approximately 30 % higher proportions of EPA (P = 0.002-0.004) and 9 % higher DHA (P = 0.013-0.017) than non-carriers. Minor allele carriers could therefore particularly benefit from a high intake of EPA and DHA in maintaining high levels of plasma n-3 PUFA conducive to protection from CVD.

Adiponectin gene variant interacts with fish oil supplementation to influence serum adiponectin in older individuals.

Marine n3 polyunsaturated fatty acids (PUFAs) activate the transcription factor peroxisome proliferator-activated receptor (PPARγ), which modulates the expression of adiponectin. We investigated the interaction of dietary n3 PUFAs with adiponectin gene (ADIPOQ) single nucleotide polymorphism (SNP) genotypes as a determinant of serum adiponectin concentration. The Modulation of Atherosclerosis Risk by Increasing Doses of n3 Fatty Acids study is a parallel design, double-blind, controlled trial. Serum adiponectin was measured in 142 healthy men and 225 women aged 45-70 y randomized to treatment with doses of 0.45, 0.9, and 1.8 g/d 20:5n3 and 22:6n3 (1.51:1), or placebo for 12 mo. The 310 participants who completed the study were genotyped for 5 SNPs at the ADIPOQ locus: -11391 G/A (rs17300539), -11377 C/G (rs266729), -10066 G/A (rs182052), +45 T/G (rs2241766), and +276 G/T (rs1501299). The -11391 A-allele was associated with a higher serum adiponectin concentration at baseline (n = 290; P < 0.001). The interaction between treatment and age as a determinant of adiponectin was significant in participants aged >58 y after the highest dose (n = 92; P = 0.020). The interaction between +45 T/G and treatment and age was a nominally significant determinant of serum adiponectin after adjustment for BMI, gender, and ethnicity (P = 0.029). Individuals homozygous for the +45 T-allele aged >58 y had a 22% increase in serum adiponectin concentration compared with baseline after the highest dose (P-treatment effect = 0.008). If substantiated in a larger sample, a diet high in n3 PUFAs may be recommended for older individuals, especially those of the +45 TT genotype who have reported increased risk of hypoadiponectinemia, type 2 diabetes, and obesity.

Genetic variation at the FADS1-FADS2 gene locus influences delta-5 desaturase activity and LC-PUFA proportions after fish oil supplement.

Delta-5 and delta-6 desaturases (D5D and D6D) are key enzymes in endogenous synthesis of long-chain PUFAs. In this sample of healthy subjects (n = 310), genotypes of single nucleotide polymorphisms (SNPs) rs174537, rs174561, and rs3834458 in the FADS1-FADS2 gene cluster were strongly associated with proportions of LC-PUFAs and desaturase activities estimated in plasma and erythrocytes. Minor allele carriage associated with decreased activities of D5D (FADS1) (5.84 × 10(-19) ≤ P ≤ 4.5 × 10(-18)) and D6D (FADS2) (6.05 × 10(-8) ≤ P ≤ 4.20 × 10(-7)) was accompanied by increased substrate and decreased product proportions (0.05 ≤ P ≤ 2.49 × 10(-16)). The significance of haplotype association with D5D activity (P = 2.19 × 10(-17)) was comparable to that of single SNPs, but haplotype association with D6D activity (P = 3.39 × 10(-28)) was much stronger. In a randomized controlled dietary intervention, increasing eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) intake significantly increased D5D (P = 4.0 × 10(-9)) and decreased D6D activity (P = 9.16 × 10(-6)) after doses of 0.45, 0.9, and 1.8 g/day for six months. Interaction of rs174537 genotype with treatment was a determinant of D5D activity estimated in plasma (P = 0.05). In conclusion, different sites at the FADS1-FADS2 locus appear to influence D5D and D6D activity, and rs174537 genotype interacts with dietary EPA+DHA to modulate D5D.

Effect of interaction between PPARG, PPARA and ADIPOQ gene variants and dietary fatty acids on plasma lipid profile and adiponectin concentration in a large intervention study.

Unsaturated fatty acids are ligands of PPAR-γ, which up-regulates genes involved in fatty acid transport and TAG synthesis and the insulin-sensitising adipokine adiponectin, which activates fatty acid ß-oxidation via PPAR-α action in liver. We investigated the effect of dietary fatty acid interaction with PPARG, PPARA and ADIPOQ gene variants on plasma lipid and adiponectin concentrations in the Reading Imperial Surrey Cambridge King's study, a five-centre, parallel design, randomised controlled trial of 466 subjects at increased cardiometabolic risk. After a 4-week run-in to baseline, SFA was replaced by MUFA or carbohydrate (low fat) in isoenergetic diets for 24 weeks. Habitual dietary PUFA:SFA ratio×PPARG Pro12Ala genotype interaction influenced plasma total cholesterol (P=0·02), LDL-cholesterol (P=0·002) and TAG (P=0·02) concentrations in White subjects. PPARA Val162Leu×PPARG Pro12Ala genotype interaction influenced total cholesterol (P=0·04) and TAG (P=0·03) concentrations at baseline. After high-MUFA and low-fat diets, total cholesterol and LDL-cholesterol were reduced (P<0·001) and gene×gene interaction determined LDL-cholesterol (P=0·003) and small dense LDL as a proportion of LDL (P=0·012). At baseline, ADIPOQ -10066 G/A A-allele was associated with lower serum adiponectin (n 360; P=0·03) in White subjects. After the high-MUFA diet, serum adiponectin increased in GG subjects and decreased in A-allele carriers (P=0·006 for difference). In GG, adiponectin increased with age after the high MUFA and decreased after the low-fat diet (P=0·003 for difference at 60 years). In conclusion, in Whites, high dietary PUFA:SFA would help to reduce plasma cholesterol and TAG in PPARG Ala12 carriers. In ADIPOQ -10066 GG homozygotes, a high-MUFA diet may help to increase adiponectin with advancing age.

Interaction of PPARG Pro12Ala with dietary fat influences plasma lipids in subjects at cardiometabolic risk.

The PPARγ2 gene single nucleotide polymorphism (SNP) Pro12Ala has shown variable association with metabolic syndrome traits in healthy subjects. The RISCK Study investigated the effect of interaction between genotype and the ratio of polyunsaturated:saturated (P:S) fatty acid intake on plasma lipids in 367 white subjects (ages 30-70 years) at increased cardiometabolic risk. Interaction was determined after habitual diet at recruitment, at baseline after a 4-week high-SFA (HS) diet, and after a 24-week reference (HS), high-MUFA (HM), or low-fat (LF) diet. At recruitment, there were no significant associations between genotype and plasma lipids; however, P:S × genotype interaction influenced plasma total cholesterol (TC) (P = 0.02), LDL-cholesterol (LDL-C) (P = 0.002), and triglyceride (TG) (P = 0.02) concentrations. At P:S ratio ≤ 0.33, mean TC and LDL-C concentrations in Ala12 allele carriers were significantly higher than in noncarriers (respectively, P = 0.003; P = 0.0001). Significant trends in reduction of plasma TC (P = 0.02) and TG (P = 0.002) concentrations occurred with increasing P:S (respectively, ≤0.33 to >0.65; 0.34 to >0.65) in Ala12 allele carriers. There were no significant differences between carriers and noncarriers after the 4-week HS diet or 24-week interventions. Plasma TC and TG concentrations in PPARG Ala12 allele carriers decrease as P:S increases, but they are not dependent on a reduction in SFA intake.

Single nucleotide polymorphisms at the ADIPOQ gene locus interact with age and dietary intake of fat to determine serum adiponectin in subjects at risk of the metabolic syndrome.

BACKGROUND: Adiponectin gene expression is modulated by peroxisome proliferator-activated receptor γ, which is a transcription factor activated by unsaturated fatty acids. OBJECTIVE: We investigated the effect of the interaction between variants at the ADIPOQ gene locus, age, sex, body mass index (BMI), ethnicity, and the replacement of dietary saturated fatty acids (SFAs) with monounsaturated fatty acids (MUFAs) or carbohydrates on serum adiponectin concentrations. DESIGN: The RISCK (Reading, Imperial, Surrey, Cambridge, and Kings) study is a parallel-design, randomized controlled trial. Serum adiponectin concentrations were measured after a 4-wk high-SFA (HS) diet and a 24-wk intervention with reference (HS), high-MUFA (HM), and low-fat (LF) diets. Single nucleotide polymorphisms at the ADIPOQ locus -11391 G/A (rs17300539), -10066 G/A (rs182052), -7734 A/C (rs16861209), and +276 G/T (rs1501299) were genotyped in 448 participants. RESULTS: In white Europeans, +276 T was associated with higher serum adiponectin concentrations (n = 340; P = 0.006) and -10066 A was associated with lower serum adiponectin concentrations (n = 360; P = 0.03), after adjustment for age, BMI, and sex. After the HM diet, -10066 G/G subjects showed a 3.8% increase (95% CI: -0.1%, 7.7%) and G/A+A/A subjects a 2.6% decrease (95% CI: -5.6%, 0.4%) in serum adiponectin (P = 0.006 for difference after adjustment for the change in BMI, age, and sex). In -10066 G/G homozygotes, serum adiponectin increased with age after the HM diet and decreased after the LF diet. CONCLUSION: In white -10066 G/G homozygotes, an HM diet may help to increase adiponectin concentrations with advancing age. This trial was registered at clinicaltrials.gov as ISRCTN29111298.

PPARγ2 gene Pro12Ala and PPARα gene Leu162Val single nucleotide polymorphisms interact with dietary intake of fat in determination of plasma lipid concentrations.

BACKGROUND/AIMS: The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of lipid metabolism, activated by unsaturated fatty acids. We investigated independent and interactive effects of PPARγ2 gene PPARG Pro12Ala (rs1801282) andPPARαgene PPARA Leu162Val (rs1800206) genotypes with dietary intake of fatty acids on concentrations of plasma lipids in subjects of whom 47.5% had metabolic syndrome. METHODS: The RISCK study is a parallel design, randomised controlled trial. Plasma lipids were quantified at baseline after a 4-week high saturated fatty acids diet and after three parallel 24-week interventions with reference (high saturated fatty acids), high monounsaturated fatty acids and low-fat diets. Single nucleotide polymorphisms were genotyped in 466 subjects. RESULTS: At baseline, the PPARG Ala12allele was associated with increased plasma total cholesterol (n = 378; p = 0.04), LDL cholesterol (p = 0.05) and apoB (p =0.05) after adjustment for age, gender and ethnicity. At baseline, PPARA Leu162Val × PPARG Pro12Ala genotype interaction did not significantly influence plasma lipid concentrations. After dietary intervention, gene-gene interaction significantly influenced LDL cholesterol (p =0.0002) and small dense LDL as a proportion of LDL (p = 0.005) after adjustments. CONCLUSIONS: Interaction between PPARG Pro12Ala and PPARA Leu162Val genotypes may influence plasma LDL cholesterol concentration and the proportion as small dense LDL after a high monounsaturated fatty acids diet.

Functional single-nucleotide polymorphism in acetyl-CoA carboxylase ACACB gene promoter.

The acetyl-CoA carboxylase isoform ACC2 expressed in the liver generates malonyl-CoA, which primarily regulates fatty acid oxidation through inhibition of the mitochondrial carrier carnitine palmitoyl-CoA transferase-I. Activity is initiated by sterol regulatory element-binding protein-1 (SREBP-1) binding to steroid response elements SRE in ACACB gene promoter P-II. We proposed that sequence variation in the promoter might affect expression. We investigated the effect of a single-nucleotide polymorphism -368 C/T (rs16939972) in ACACB P-II on activity in transfected HepG2 hepatoma cells. The T-allele construct showed significantly lower activity than the C-allele (p = 0.016) but only in the presence of SREBP-1a overexpression. Electrophoretic mobility shift assays showed that HepG2 nuclear proteins bound specifically to both allele probes, but with higher affinity to the T-allele. We tested competition for nuclear protein binding between the T-allele probe and unlabeled probes containing consensus sequences for six candidate transcription factors plus SREBP-1a. The SREBP-1a competitor probe had no effect on the shifted complex. GATA, c-Myb, and GR competitor probes abolished the complex; however, these proteins were undetectable in mass spectrometry of gel extracts from shifted bands. In conclusion, the -368 C/T single-nucleotide polymorphism in ACACB P-II binds HepG2 nuclear proteins that affect promoter activity in an allele-specific fashion.

Effect of high-fat feeding on expression of genes controlling availability of dopamine in mouse hypothalamus.

OBJECTIVE: Hypothalamic centers integrate external signals of nutrient availability and energy status and initiate responses to maintain homeostasis. Quantifying changes in hypothalamic gene expression in the presence of nutrient excess may identify novel responsive elements. METHODS: Affymetrix Mouse Genome 430 2.0 oligonucleotide microarrays containing 45 102 probe sets were used to interrogate differential expression of genes in dietary-induced obesity model C57BL6 inbred mice fed a high-fat (35% fat; n=8) or standard (4% fat; n=6) diet from 3 to 15 wk of age. Ontologies of regulated genes were examined and expression of selected genes was validated by quantitative real-time polymerase chain reaction. RESULTS: One thousand two hundred twelve unique gene transcripts showed altered expression on the microarrays. Gene ontology analysis revealed changes in neuropeptide genes responding to leptin, Pomc, Cart, Npy, and Agrp, compatible with a homeostatic response to high-fat intake, although mean weight increased 2.3-fold compared with standard fed mice (P<0.001). Neurotransmitter system ontologies revealed upregulation of five genes controlling availability of dopamine. Changes in Th tyrosine hydroxylase (2.1-fold) and Slc18a2 solute carrier family 18 (vesicular monoamine), member 2 (4.4-fold) controlling synthesis and release, and Slc6a3 solute carrier family 6 (neurotransmitter transporter, dopamine), member 3 (4.8-fold), Snca alpha-synuclein (1.3-fold), and Maoa monoamine oxidase (1.9-fold) limiting availability were confirmed by quantitative real-time polymerase chain reaction. CONCLUSION: Expression of five genes involved in availability of dopamine was increased after a high-fat diet. Failure to reduce dopamine availability sufficiently, to counter the feeding reward effect, could contribute to diet-induced obesity in these mice.

CD36 gene promoter polymorphisms are associated with low density lipoprotein-cholesterol in normal twins and after a low-calorie diet in obese subjects.

Common polymorphisms of the CD36 fatty acid transporter gene have been associated with lipid metabolism and cardiovascular disease. Association of a CD36 promoter single nucleotide polymorphism genotype with anthropometry and serum lipids was investigated in normal subjects, and in obese subjects during an 8-week low calorie diet and 6-month weight-maintenance period. 2728 normal female Twins UK subjects (mean body mass index 24.8 +/- 4.4 kg/m2; age 47.3 +/- 12.5 y) and 183 obese male and female Spanish subjects (mean body mass index 30.6 +/- 3.0 kg/m2; age 35.0 +/- 5.0 y) were genotyped for the CD36-22674 T/C (rs2151916) promoter single nucleotide polymorphism. In the Twins UK full cohort, the C-allele was associated with lower low density lipoprotein-cholesterol (p = .02, N = 2396). No associations were found in the obese Spanish subjects at baseline, but 6 months after the end of the low-calorie diet, the C-allele was associated with lower total- (p = .03) and low density lipoprotein-cholesterol (p = .01) and higher high density lipoprotein-cholesterol (p = .01). Intake of saturated fatty acids was lower in carriers of the C-allele at baseline, but not significantly so (p = .11). However, 6 months after the end of the low-calorie diet, elements of the lipid profile were correlated with saturated fatty acid intake: total cholesterol r = .21, p = .060; low density lipoprotein-cholesterol: r = .25, p = .043; high density lipoprotein-cholesterol: r = -.26, p = .007. CD36 promoter SNP allele -22674C is therefore associated with lower serum low-density lipoprotein-cholesterol in normal female twins and with improved lipid profile during weight loss and maintenance in obese subjects.

Adiponectin gene ADIPOQ SNP associations with serum adiponectin in two female populations and effects of SNPs on promoter activity.

Adiponectin is an insulin sensitiser in muscle and liver, and low serum levels characterise obesity and insulin resistance. Eight tagging single nucleotide polymorphisms (tSNPs) in the ADIPOQ gene and promoter were selected, and association with serum adiponectin was tested, in two independent samples of Caucasian women: the Chingford Study (n = 808, mean age 62.8 +/- 5.9 years) and Twins UK (n = 2,718, mean age 47.4 +/- 12.6 years). In the Chingford cohort, -11391 G/A, -10066 G/A (rs182052), -7734 C/A (rs16861209), +276 G/T (rs1501299) and +3228 C/T (rs1063537) were significantly associated with fasting serum adiponectin (Ps = 1.00 x 10(-4) to 1.40 x 10(-2)). Associations with all except +3228 C/T were replicated in the Twins UK cohort (Ps = 3.19 x 10(-9) to 6.00 x 10(-3)). In Chingford subjects, the 12 most common 8-SNP haplotypes (frequency 1.90%) explained 2.85% (p = 5.00 x 10(-2)) and in Twins UK subjects, the four most common 5-SNP haplotypes (frequency > 5.00%) explained 1.66% of the variance (p = 5.83 x 10(-7)). To investigate effects of -11391 G/A (rs17300539) and -11377 C/G (rs266729) on promoter activity, 1.2 kb of the ADIPOQ promoter region was cloned in a luciferase reporter plasmid, and the four haplotypes were transfected in differentiated 3T3-L1 adipocytes. No significant allelic effects on promoter activity were found.

Association of common JAK2 variants with body fat, insulin sensitivity and lipid profile.

The leptin signal is transduced via the JAK2-STAT3 (Janus kinase 2-signal transducer and activator of transcription-3) pathway at the leptin receptor. JAK2 also phosphorylates insulin receptor substrate, integral to insulin and leptin action and is required for optimum adenosine triphosphate-binding cassette transporter A1 (ABCA1)-dependent transport of lipids from cells to apolipoprotein A-1 (apoA-I). We hypothesized that common variation in the JAK2 gene may be associated with body fat, insulin sensitivity, and modulation of the serum lipid profile in the general population. Ten tagging single-nucleotide polymorphisms (SNPs) spanning the gene were genotyped in 2,760 white female twin subjects (mean age 47.3 +/- 12.6 years) from the St Thomas' UK Adult Twin Registry. Minor allele frequencies were between 0.170 and 0.464. The major allele of rs7849191 was associated with higher central fat (P = 0.030), percentage of central fat (P = 0.014) and waist circumference (P = 0.027) the major allele of rs3780378 with higher serum apoA (P = 0.026), total cholesterol (P = 0.014), low-density lipoprotein (LDL) cholesterol (P = 0.012) and lower triglyceride (P = 0.023). However, no associations were significant at a level which took account of multiple testing. Although JAK2 is a critical element in leptin and insulin signaling and has a role in cellular cholesterol transport, we failed to establish associations of common SNPs with relevant phenotypes in this human study.

Common STAT3 variants are not associated with obesity or insulin resistance in female twins.

In animal models, STAT3 action in the hypothalamus and liver appears essential for normal body weight and glucose homeostasis in response to insulin. We hypothesized that variation in the STAT3 gene may be associated with body fat and/or insulin resistance in the general population. Five tagging SNPs spanning the STAT3 gene, rs8074524, rs2293152, rs2306580, rs6503695, and rs7211777 were genotyped in 2776 white female twins (mean age, 47.4+/-12.5 yrs) from the St Thomas' United Kingdom Adult Twin Registry (Twins UK). Minor allele frequencies were as follows: rs8074524 (0.19), rs2293152 (0.37), rs2306580 (0.06), rs6503695 (0.35), and rs7211777 (0.34). The minor allele of rs2293152 was associated with higher homeostasis model assessment index of insulin resistance (p=0.013) in the full cohort and confirmed in sib-transmission/disequilibrium test (TDT): (p=0.015; n=60). However, there were no associations with fasting serum insulin or glucose or with obesity variables. Although defective STAT3 action results in obesity and insulin resistance in animal models, we failed to establish any indicative associations with common SNPs in this human study.

The SH2B gene is associated with serum leptin and body fat in normal female twins.

Src-homology-2 (SH2)-B, a Janus tyrosine kinase 2-interacting protein, has been identified recently as a key regulator of leptin and insulin sensitivity, glucose homeostasis, and body weight in mice. The aim of this study was to determine whether single-nucleotide polymorphisms (SNPs) in the human SH2B gene are associated with these variables. A tagging SNP (tSNP), Ala484Thr (rs7498665), was selected to represent five common SNPs (minor allele frequency > 0.05) in perfect linkage disequilibrium in a 16-kb region encompassing the SH2B gene. The tSNP was genotyped in 2455 white female twins (mean age, 47.4 +/- 12.6 years) from the St. Thomas' United Kingdom Adult Twin Registry (Twins United Kingdom). Ala484Thr (minor allele frequency, 0.38) was associated with serum leptin, total fat, waist circumference, and body weight (P = 0.02 to 0.04). The coding SNP has no predicted effect on protein structure or function and is likely to be in linkage disequilibrium with an as-yet unidentified functional variant in the SH2B gene. Our results support a role for SH2-B in modulating the regulation of body weight and fat by leptin in this female population. If SH2-B signaling is attenuated in diet-induced obesity, it could become a target for drug-induced leptin sensitization.

Prevalence and functionality of paucimorphic and private MC4R mutations in a large, unselected European British population, scanned by meltMADGE.

Identification of unknown mutations has remained laborious, expensive, and only viable for studies of selected cases. Population-based "reference ranges" of rarer sequence diversity are not available. However, the research and diagnostic interpretation of sequence variants depends on such information. Additionally, this is the only way to determine prevalence of severe, moderate, and silent mutations and is also relevant to the development of screening programs. We previously described a system, meltMADGE, suitable for mutation scanning at the population level. Here we describe its application to a population-based study of MC4R (melanocortin 4 receptor) mutations, which are associated with obesity. We developed nine assays representing MC4R and examined a population sample of 1,100 subjects. Two "paucimorphisms" were identified (c.307G>A/p.Val103Ile in 27 subjects and c.-178A>C in 22 subjects). Neither exhibited any anthropometric effects, whereas there would have been >90% power to detect a body mass index (BMI) effect of 0.5 kg/m(2) at P=0.01. Two "private" variants were also identified. c.335C>T/p.Thr112Met has been previously described and appears to be silent. A novel variant, c.260C>A/p.Ala87Asp, was observed in a subject with a BMI of 31.5 kg/m(2) (i.e., clinically obese) but not on direct assay of a further 3,525 subjects. This mutation was predicted to be deleterious and analysis using a cyclic AMP (cAMP) responsive luciferase reporter assay showed substantial loss of function of the mutant receptor. This population-based mutation scan of MC4R suggests that there is no severe MC4R mutation with high prevalence in the United Kingdom, but that obesity-causing MC4R mutation at 1 in 1,100 might represent one of the commonest autosomal dominant disorders in man.

Protein tyrosine phosphatase-1B gene PTPN1: selection of tagging single nucleotide polymorphisms and association with body fat, insulin sensitivity, and the metabolic syndrome in a normal female population.

Protein tyrosine phosphatase-1B negatively regulates leptin and insulin signaling, potentially contributing to hormonal resistance. We selected six tagging single nucleotide polymorphisms (SNPs) representing 18 common variants in the protein tyrosine phosphatase-1B gene (PTPN1) and tested their effect on serum leptin, body fat, and measures of insulin sensitivity and the metabolic syndrome in a large sample of normal female Caucasian twins (n = 2,777; mean age, 47.4 +/- 12.5 years) from the St. Thomas' U.K. Adult Twin Registry. SNP rs718049 was significantly associated with waist circumference (P = 0.008) and central fat (P = 0.035) and also with Avignon's insulin sensitivity index (SiM) (P = 0.007), fasting insulin (P = 0.004), fasting glucose (P = 0.022), triglyceride (P = 0.023), and systolic blood pressure (P = 0.046). SNPs rs2282146 and rs1885177 were associated with SiM (P = 0.049 and P = 0.013, respectively), and 1484insG was associated with triglyceride (P = 0.029). A risk haplotype (7.3%) was associated with lower SiM (P = 0.036) and a protective haplotype (5.2%) with higher SiM (P = 0.057), with mean values in homozygotes differing by >1 SD (P = 0.003). The protective haplotype also showed lower triglyceride (P = 0.045) and lower systolic blood pressure (P = 0.006). Fine mapping analyses predicted significant associations with SiM and fasting insulin for several ungenotyped SNPs. PTPN1 variants appear to contribute to central fat and metabolic syndrome traits, secondary to their effect on insulin sensitivity.