The Val1483Ile polymorphism in the human fatty acid synthase (FASN) gene is located within the interdomain region of the FASN close to the two dynamic active centers of the FASN enzyme and putatively affects FASN action. We aimed to evaluate the association of this polymorphism with obesity phenotypes, insulin sensitivity, and adipose tissue FASN activity in adult white subjects. The polymorphism was evaluated in association with metabolic variables in two independent studies: in a case–control study of 457 men (229 with normal glucose tolerance (NGT) and 228 with altered glucose tolerance (AGT)); and in 600 population-based NGT subjects (274 men and 326 women). Adipose tissue FASN activity was analyzed using the method of Nepokroeff. The Ile variant was associated with a lower waist-to-hip ratio (WHR) and a lower increase in weight over a 7-year period in NGT men. In a subset of 147 men, carriers of the Ile variant showed significantly increased insulin sensitivity. BMI (P < 0.001), WHR (P = 0.03), and Val1483Ile (P = 0.03), contributed independently to 37% of insulin sensitivity variance. In men from the population-based study, the Ile variant was associated with a lower BMI, WHR, fasting glucose, and systolic blood pressure compared with carriers of the Val variant. In agreement with these results, the adipose tissue FASN activity was significantly lower in subjects with the Ile variant (P = 0.01). In summary, adult white men with the Ile 1483 variant of the FASN gene seem protected from developing central obesity through decreased adipose tissue FASN activity.
Obesity and associated comorbidities are the most common and detrimental metabolic diseases, affecting over 50% of adult population. Adiposity is closely linked to important physiological parameters, such as blood pressure, systemic insulin sensitivity, and fasting triglyceride concentrations (1,2). Visceral fat seems more closely correlated with obesity-associated disorders than overall adiposity (3,4), and is well known to constitute an independent risk factor for myocardial infarction, stroke, type 2 diabetes mellitus, and certain cancers (5,6). In this sense, the waist-to-hip ratio (WHR) has been shown to be a stronger predictor of cardiovascular events than BMI (7). The search for genes that increase the susceptibility to develop central obesity or that interact with multiple pathophysiological mechanisms of energy homeostasis has become increasingly important (8). One of these genes is human fatty acid synthase (FASN) gene.
The human FASN gene codes for an enzyme essential for de novo fatty acid synthesis and cellular substrate energy metabolism. Active FASN is a homodimer in which each peptide subunit has a molecular weight of 260 kDa. Loftus and co-workers showed that inhibition of FASN induced a rapid decline in fat stores in mice, suggesting an implication for this enzyme in energy homeostasis (9). In fact, FASN gene expression is upregulated by excess energy intake and increased insulin levels (10,11,12). Excessive FASN activity in visceral adipose tissue could lead to an increase in the concentration of fatty acids in the portal vein, altering hepatic metabolism and promoting insulin resistance (13). Interestingly, increased FASN gene expression in visceral adipose tissue has been linked to impaired insulin sensitivity and increased circulating adipokines, such as interleukin-6 and leptin (14).
The FASN gene is positioned on chromosome 17q25 within a region of linkage with body fat content in Pima Indians (15). Kovacs et al. reported a novel missense mutation in the FASN gene, which was significantly associated with body fat content and substrate oxidation rate in Pima Indians (16). This gene variant was also associated with a protective effect regarding the development of obesity in white boys but not in girls (17). This FASN gene polymorphism leads to a change of Val for Ile in the 1483 position of the amino acid sequence. This amino acid is positioned within the interdomain region of the FASN molecule that is close to two dynamic active centers of the FASN dimmer (18). For this reason, this change in the structure of the FASN protein may alter the configuration of the catalytically active enzyme and thus, putatively, alter its biological activity. However, no study has evaluated this possibility in vivo.
The aim of this study was thus to evaluate whether this polymorphism is associated with adiposity phenotypes and insulin resistance in adult white subjects and to characterize adipose tissue FASN activity according to this FASN gene Val1483Ile polymorphism.
Methods and Procedures
A total of 457 white men were recruited and studied. Of them, 343 were recruited in ongoing study dealing with nonclassical cardiovascular risk factors in Northern Spain. Subjects were randomly localized from a census and they were invited to participate. The participation rate was 71%. A 75-g oral glucose tolerance test according to the American Diabetes Association Criteria was performed in all subjects. All subjects with normal glucose tolerance (NGT; n = 229) had fasting plasma glucose <7.0 mmol/l and 2-h postload plasma glucose <7.8 mmol/l after a 75-g oral glucose tolerance test. Impaired glucose tolerance was diagnosed in 88 subjects according to the American Diabetes Association Criteria (postload glucose between 7.8 and 11.1 mmol/l). Previously unknown type 2 diabetes was diagnosed in 26 of these 343 subjects (postload glucose >11.1 mmol/l).
Inclusion criteria were (i) BMI <40 kg/m2, (ii) absence of systemic disease, and (iii) absence of infection within the previous month, and (iv) serum ferritin >10 ng/ml and normal blood hemoglobin concentration (>12 g/dl) to exclude iron deficiency. None of the control subjects were under medication or had evidence of metabolic disease other than obesity. Alcohol and caffeine were withheld within 12 h before performing the oral glucose tolerance test. Smokers were defined as any person consuming at least one cigarette a day in the previous 6 months. Liver disease and thyroid dysfunction were specifically excluded by biochemical workup.
To increase the statistical power of the group of patients with type 2 diabetes, 114 patients were prospectively recruited from diabetes outpatient clinics on the basis of a stable metabolic control in the previous 6 months, as defined by stable HbA1c values. Data from these patients were merged with those with recently diagnosed type 2 diabetes. Exclusion criteria for these patients included the following: (i) clinically significant hepatic, neurological, endocrinologic, or other major systemic disease, including malignancy; (ii) history or current clinical evidence of hemochromatosis; (iii) history of drug or alcohol abuse, defined as >80 g/day in men and >40 g/day in women, or serum transaminase activity more than twice the upper limit of normal; (iv) an elevated serum creatinine concentration; (v) acute major cardiovascular event in the previous 6 months; (vi) acute illnesses and current evidence of acute or chronic inflammatory or infective diseases; and (vii) mental illness rendering the subjects unable to understand the nature, scope, and possible consequences of the study. Pharmacological treatment for these patients was insulin: 31 patients; metformin: 37 patients; sulfonylureas, 21 patients; statins: 44 patients; fibrates: 9 patients; blood pressure–lowering agents: 41 patients; aspirin: 49 patients; and allopurinol: 3 patients. All subjects gave written informed consent after the purpose of the study was explained to them. The institutional review board of the institution approved the protocol.
Subjects were studied in the postabsorptive state. BMI was calculated as weight (in kilograms) divided by height (in meters squared). Subjects' waists were measured using a soft tape midway between the lowest rib and the iliac crest; hip circumference was measured at the widest part of the gluteus region; and WHR was accordingly calculated. Blood pressure was measured in the supine position on the right arm after a 10-min rest; a standard sphygmomanometer of appropriate cuff size was used and the first and fifth phases were recorded. Values used in the analysis are the average of three readings taken at 5-min intervals.
Insulin sensitivity was measured using the frequently sampled intravenous glucose tolerance test on a different day in those subjects who agreed (n = 147). In brief, basal blood samples were drawn at −15 and −5 min, after which glucose (300 mg/kg body weight) was injected over 1 min starting at time 0. At 20 min, regular insulin (Actrapid, 0.03 U/kg; Novo Nordisk, Bagsvaerd, Denmark) was injected as a bolus. Additional samples were obtained from a contralateral antecubital vein at times 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 19, 20, 22, 23, 24, 25, 27, 30, 40, 50, 60,70, 80, 90, 100, 120, 140, 160, and 180 min. Samples were rapidly collected using a three-way stopcock connected to the butterfly needle. Data from the frequently sampled intravenous glucose tolerance test were submitted to computer programs that calculate the characteristic metabolic parameters by fitting glucose and insulin to the minimal model that describes the times course of glucose and insulin concentrations. The glucose disappearance model, by accounting for the effect of insulin and glucose on glucose disappearance, provides the parameters SI (10−4 per minute per microunit per milliliter) or the insulin sensitivity index, a measure of the effect of insulin concentrations above the basal level to enhance glucose disappearance. The estimation of model parameters was performed according to the MINMOD computer program (19).
Participants were 696 consecutive apparently healthy subjects enrolled in a cross-sectional, population-based study examining the prevalence of cardiovascular risk factors in Northwestern Spain (20). All subjects were of white origin and reported that their body weight had been stable for at least 3 months before the study. Inclusion criteria for this group were (i) BMI <40 kg/m2, (ii) absence of any systemic disease, (iii) alcohol intake <40 g/day in men and 20 g/day in women. Subjects underwent an oral glucose tolerance test to diagnose type 2 diabetes, according to American Diabetes Association criteria, if this disease had not been previously diagnosed according to these guidelines (21). Newly diagnosed type 2 diabetic patients and those with previously diagnosed disease (n = 96) were not included in this study.
Men (n = 274) were characterized by being more centrally obese, hyperglycemic, hypertriglyceridemic, and hypertensive than women (n = 326) (Table 3). They were studied separately in the genotype–phenotype association analyses.
Table 3. Anthropometrical and biochemical variables in the population-based study
Informed written consent was obtained after the purpose, nature, and potential risks of the study were explained to the subjects. The experimental protocol was approved by the Ethics Committee of the Hospital of Asturias.
In this study, insulin resistance was measured by the homeostasis model assessment (HOMA) of insulin resistance (22). It is well known that HOMA correlates well with insulin sensitivity derived from the glucose clamp technique (r = −0.82, P < 0.0001), and this correlation appears to be independent of sex, age, BMI, diabetes, and blood pressure (23).
Serum glucose concentrations were measured in duplicate by the glucose oxidase method using a Beckman glucose analyzer II (Beckman Instruments, Brea, CA). High-density lipoprotein (HDL) cholesterol was quantified after precipitation with polyethylene glycol at room temperature. Total serum triglycerides were measured through the reaction of glycerol-phosphate-oxidase and peroxidase on a Hitachi 917 instrument (Roche, Mannheim, Germany). Serum insulin was measured in duplicate in the same centralized laboratory by monoclonal immunoradiometric assay (Medgenix Diagnostics, Fleunes, Belgium). The intra-assay coefficient of variation was 5.2% at a concentration of 10 mU/l and 3.4% at 130 mU/l. The interassay coefficient of variations were 6.9 and 4.5% at 14 and 89 mU/l, respectively.
Single-nucleotide polymorphism analysis
Genomic DNA was extracted from peripheral blood leukocytes according to standard procedures (QIAamp DNA Blood Mini Kit; Qiagen, Hilden, Germany) and quantified using a spectrophotometer (GeneQuant; GE Health Care, Piscataway, NJ). In total, 100 ng of DNA were used for PCR amplification. For the detection of the polymorphisms, iPLEXTM chemistry on a MALDI-TOF Mass Spectrometer (Sequenom, San Diego, CA) was used. To validate the reproducibility of the method, 20 samples were regenotyped and the results were 100% identical. The four groups of population studied were in Hardy–Weinberg equilibrium for this single-nucleotide polymorphism (SNP) (χ2 = 0.04, P = 0.97 in control subjects, and χ2 = 0.015, P = 0.99 in subjects with altered glucose tolerance (AGT) of the case–control study; χ2 = 0.005, P = 0.99 in men and χ2 = 0.005, P = 0.99 in women from the population-based study).
FASN activity assay
Six samples of adipose tissue were obtained from obese subjects (three with Ile/Val and the other three with Val/Val variant) undergoing open abdominal surgery (gastrointestinal bypass) under anesthesia after an overnight fast. Subjects with Ile/Val and Val/Val variants were similar in age (57 ± 10.1 vs. 53.3 ± 2.5 years, respectively, P = 0.57), BMI (36.1 ± 9.2 kg/m2 vs. 31.2 ± 2.6 kg/m2, P = 0.43), fasting glucose (4.8 ± 0.35 mmol/l vs. 4.27 ± 0.62 mmol/l, P = 0.23), and HOMA value (2.72 ± 1.6 vs. 1.4 ± 0.7, P = 0.45). Medical histories, physical examination, electrocardiogram, and analytical tests showed that all patients were in good health.
Frozen adipose subcutaneous tissue was homogenized at 37 °C in a 10 mmol/l HEPES buffer containing 0.25 mol/l sucrose, 1 mmol/l EDTA, and 1 mmol/l dithiothreitol. The homogenate was centrifuged at 13,000g at 0–4 °C for 30 min. The floating fat layer was removed and aliquots of the fluid infranatant fraction were taken for determination of FASN activity (24). The protein concentration of the fluid infranatant fraction was determined by the Lowry assay.
FASN activity was determined using the method of Nepokroeff (25). Aliquots of the tissue extracts were incubated at 37 °C for 15 min with phosphate buffer (1 mmol/l potassium phosphate, 0.2 mmol/l EDTA, and 1 mol/l dithiothreitol, pH = 7.0). After addition of nicotinamide adenine dinucleotide phosphate (NADPH) (100 µmol/l final concentration), the reaction was initiated by adding acetyl- and malonyl-CoA (35 µmol/l and 100 µmol/l final concentration, respectively). The reaction was performed with 50 µg of protein. The change in absorbance at 340 nm due to oxidation of NADPH was followed with a Zenyth 200 spectrophotometer at 37 °C for 10 min. A unit of FASN activity is defined as 1 nmol of NADPH oxidized/min.
We also performed a western blot using 50 µg of protein to test the FASN quantity in the sample. Protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes by conventional procedures. Membranes were immunoblotted with FASN antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Anti-rabbit IgG coupled to horseradish peroxidase was used as secondary antibody. Horseradish peroxidase activity was detected by chemiluminescence, and quantification of protein expression was performed using Scion image software.
Statistical analyses were performed using SPSS 12.0 software. Unless otherwise stated, descriptive results of continuous variables are expressed as mean and s.d. for Gaussian variables and median and interquartile range for non-Gaussian variables. Parameters that did not fulfill normal distribution were log transformed to improve symmetry for subsequent analyses. Unpaired t-tests were used to compare GG/GG (Val/Val) and AG/GG (Ile/Val) genotypes. The relation between variables was analyzed by multiple linear regression analysis in a stepwise manner. The FASN gene polymorphism was entered as 1,2. Levels of statistical significance were set at P < 0.05.
Allelic frequency was different according to glucose tolerance. This SNP was more frequent in subjects with NGT than in subjects with AGT (88 subjects with impaired glucose tolerance and 140 subjects with type 2 diabetes) (0.041 vs. 0.026 for the Ile variant, respectively). Val1483Ile polymorphism was significantly linked to WHR (P = 0.02) and to an increase in weight over a 7-year period (P = 0.02) in all men as a whole and in the subset of subjects in whom insulin sensitivity was determined (Table 1).
Table 1. Comparisons of metabolic variables according to FASN gene polymorphism in the case–control study
In subjects with NGT, the Ile variant was significantly associated with a lower WHR, a lower increase in weight over a 7-year period and a higher HDL-cholesterol concentration. In AGT subjects, the Val1483Ile polymorphism was not significantly associated with metabolic variables (Table 1).
In all subjects as a whole and in subjects with NGT, the Ile variant was significantly associated with increased insulin sensitivity (Table 1). However, insulin sensitivity was available in a reduced sample of AGT subjects, which precludes reaching to any conclusion.
Finally, we performed multiple linear regression analysis to predict insulin sensitivity and HDL-cholesterol. We considered as independent variables those with significant association on univariant analysis. BMI (P < 0.001), WHR (P = 0.03), and FASN gene polymorphism (P = 0.03), contributed independently to 37% (adjusted R2) of insulin sensitivity variance, after controlling for the effects of age in all subjects as a whole (Table 2). Age (P = 0.019), WHR (P = 0.024), and FASN gene polymorphism (P = 0.032), contributed independently to 14% (adjusted R2) of HDL-cholesterol variance, after controlling for the effects of the BMI in subjects with NGT (Table 2).
Table 2. Multiple linear regression analysis in the case–control study
The main characteristics are shown in Table 3. Among men, subjects carrying the Ile variant had significantly lower BMI, WHR, fasting glucose, and systolic blood pressure, even after controlling for age. However, the associations with fasting glucose and systolic blood pressure were no longer significant after controlling for BMI or WHR in multiple linear regression models. In contrast to men, no association was seen between Val1483Ile polymorphism and metabolic parameters among women (Table 4).
Table 4. Comparisons of metabolic variables according to FASN gene polymorphism in the population-based study
Study of FASN activity in adipose tissue
FASN activity from subjects carrying the Ile/Val was significantly lower, as demonstrated by significantly lower NADPH oxidized rate, than in those samples from subjects carrying the Val/Val variant (0.006 ± 0.005 vs. 0.032 ± 0.009 nmol/min, P = 0.01, Figure 1). Western blot results showed that there were no significant differences in FASN protein among samples (Figure 1).
The Val1483Ile polymorphism has been previously investigated in Pima Indians (16) and in white children (17). This is the first study, to our knowledge, showing that the Val 1483 Ile polymorphism of the FASN gene was associated with adiposity (mainly central obesity) among adult white men with NGT. The association of the SNP with BMI was not consistently significant in the two populations. Because the association with WHR was consistent in both populations, these findings imply that this SNP was associated only with visceral obesity.
The allelic frequency of the Ile variant in the case–control study tended to be higher in subjects with NGT (0.041) compared to AGT subjects (0.026). In other white populations, the Ile variant allele frequency was 0.03 (ref. 17) that is similar to that found in the two studies reported here (0.033 and 0.0285). Interestingly, the Ile variant was associated with a lower WHR, a lower increase in weight, and increased insulin sensitivity in all subjects as a whole and with increased HDL-cholesterol in subjects with NGT. The latter associations persisted significant after controlling for adiposity and age (Table 2).
In subjects with AGT, the absence of differences in adiposity according to Val 1483 Ile polymorphism could be attributed to the concomitant treatment of these subjects (statins, fibrates, insulin, and oral antidiabetic drugs). Even after controlling for these factors, no significant differences were found. It could be argued that the heterogeneity of subjects with AGT, their treatment, and the lower allelic frequency of Ile variant could be behind this absence of associations in AGT subjects.
In the population-based study, the significant findings (lower BMI and WHR) were observed only in men. HOMA value tended to be lower in subjects carrying the Ile variant, but this finding did not reach statistical significance. The associations with fasting glucose, HOMA value, and systolic blood pressure were lost after controlling for BMI, an important contributor to the variance of these parameters. We did not observe associations between Val1483Ile polymorphism and metabolic variables among women. This study not only replicates association of the Val1483Ile with obesity, but also supports its sex-specific effects. A similar sexual dimorphism in the association between this polymorphism and adiposity and metabolic parameters (HDL-cholesterol and fasting triglycerides) has been described in white children (17). Subjects with the Val1483Ile polymorphism in the FASN gene seem protected from developing visceral obesity in both studies.
The amino acid change positioned within interdomain region of the FASN protein is close to the two dynamic active centers of the FASN dimmer. This change could modify the configuration of catalytically active FASN and reduce its activity (18,26). We assayed the FASN activity by measuring the NADPH oxidation rate using the method of Nepokroeff (24,25). We found that FASN activity was significantly decreased (by 81.2%) in adipose tissue samples from subjects carrying the Ile variant. A decrease of FASN activity using C75 (a known blocker of fatty acid synthase) was linked to improve energy utilization, decrease fatty liver and adipose tissue mass, and a higher fatty acid oxidation (27). It is thus reasonable to suggest that the lower total and central adiposity and the increased insulin sensitivity in subjects carrying the Ile variant could be due to decreased FASN activity.
Berndt et al. have recently reported that FASN expression was linked with visceral fat accumulation, increased circulating adipokines, and impaired insulin sensitivity (14). FASN activity interferes in cell cycle control and downregulates the cyclin-dependent kinase inhibitor p27Kip1. As a consequence, FASN has been proposed to promote adipocyte hyperplasia and obesity and cancer development (28,29).
In summary, the Val 1483 Ile polymorphism of the FASN gene was associated with central obesity in two cohorts of adult white subjects. This association could be due to decreased FASN activity among subjects carrying the Ile variant.
This work was partially supported by research grants from the Ministerio de Educación y Ciencia (SAF2008-0273). CIBEROBN Fisiopatología de la Obesidad y Nutrición is an initiative from the Instituto de Salud Carlos III from Spain. We acknowledge the administrative help of Paula Otero.