European Review for Medical and Pharmacological Sciences 2010; 14: 89-95 Influence of Ala54Thr polymorphism of fatty acid-binding 2 on insulin resistance and adipocytokines in patients with diabetes mellitus type 2

D.A. DE LUIS, M. GONZALEZ SAGRADO, R. ALLER, O. IZAOLA, R. CONDE, B. DE LA FUENTE

Institute of Endocrinology and Nutrition, Medicine School and Unit of Investigation, Hospital Rio Hortega, University of Valladolid, Valladolid (Spain)

Abstract. – Background: A transition G to Key Words: A at codon 54 of FABP2 results in an amino acid Adipokines, Diabetes Mellitus, FABP2, Polymor- substitution (ala 54 to Thr 54). This polymor- phism, Insulin resistance. phism could be associated with insulin resis- tance and adipocytokines in patients with dia- betes mellitus type 2. Objective: The aim of our study was to inves- tigate the influence of Thr54 polymorphism in the FABP2 on adipocytokines and insulin Introduction resistance in the fasted state in naïve patients with diabetes mellitus type 2. The current view of adipose tissue is that of an Design: A population of 58 naïve patients active secretory organ, sending out and respond- with diabetes mellitus type 2 (fasting glucose ing to signals that modulate appetite, insulin sen- >126 mg/dl) and obesity (body mass index >30) sitivity, energy expenditure, inflammation and was analyzed in a prospective way. An indirect immunity1-10. calorimetry, tetrapolar electrical bioimpedance, The fatty acid (FA) binding protein 2(FABP2) blood pressure, a serial assessment of nutri- tional intake with 3 days written food records gene codes for intestinal FABP, which is a mem- and biochemical analysis (lipid profile, adipocy- ber of a family of small intracellular lipid-bind- tokines, insulin, CRP and HOMA) were per- ing . FABP plays an important role in formed. The statistical analysis was performed several steps of unsaturated and saturated long for the combined Ala54/Thr54 and Thr54/Thr54 chain fatty acids (LCFAs), protection of the cell as a mutant group and wild type Ala54/Ala54 as from the cytotoxic effects of FFAs, and modula- second group. tion of the enzyme additive involved in lipid me- Results: Thirty-three patients (56.9%) had tabolism11-12. Recently, a group13 reported a new genotype Ala54/Ala54 (wild type group) and 25 (43.1%) patients Ala54/Thr54 (19 patients, G/A mutation. A transition G to A at codon 54 of 32.8%) or Thr54/Thr54 (6 patients, 10.3%) (mu- FABP2 results in an amino acid substitution (Ala tant group). C-reactive protein (5.6±4.2 vs 54 to Thr 54). This polymorphism is common, 8.7±5.3 mg/dl: p<0.05), insulin (22.9±11 vs with a Thr54 allelic frequency of 30% in most 6.6±12 mUI/L: p<0.05) and HOMA (7.65±5.3 vs populations. This amino acid substitution was as- 9.1±6.3: p<0.05) were higher in mutant group sociated with high insulin resistance, and fasting than wild group. Anthropometric parameters, insulin concentrations13. Carriers of the Thr54 al- dietary intakes and adipocytokines were similar in both genotypes. lele have a 2-fold greater affinity for the long- Conclusion: The novel finding of this study is chain fatty acids than those with the Ala 54, the association of the Thr54/Ala54 and Thr54/ which supports the role of the FABP2 Ala54Thr Thr54 FABP2 genotypes with higher levels of C polymorphism in the etiology of metabolic disor- reactive protein, insulin and HOMA. ders such as insulin resistance14.

Corresponding Author: Daniel A. de Luis, MD; e-mail: [email protected] 89 D.A. de Luis, M. Gonzalez Sagrado, R. Aller, O. Izaola, R. Conde, B. de La Fuente

The aim of our study was to investigate the in- ATC AAG CAC TTT TCG AAA CA-BHQ-1-3’) fluence of Thr54 polymorphism in the FABP2 and (mutant probe: 5’-Hex-AGA ATC AAG gene on adipocytokines and insulin resistance in CGC TTT TCG AAA CA-BHQ-1-3’) in a 25 µL the fasted state in naïve patients with diabetes final volume (Termociclador iCycler IQ (Bio- mellitus type 2. Rad®), Hercules, CA). DNA was denaturated at 95ºC for 3 min; this was followed by 50 cycles of denaturation at 95ºC for 15 s, and annealing at 59.3ºC for 45 s). The PCR were run in a 25 µL fi- Subjects and Methods nal volume containing 12.5 µL of IQTM Super- mix (Bio-Rad®, Hercules, CA) with hot start Taq Subjects DNA polymerase. Hardy Weimberger equilibri- A population of 58 naïve patients with dia- um was assessed. betes mellitus type 2 (fasting glucose >126 mg/dl) and obesity (body mass index >30) was Assays analyzed in a prospective way (research protocol Serum total cholesterol and triglyceride con- accepted by the Ethical Committee). These pa- centrations were determined by enzymatic col- tients were recruited in a Nutrition Clinic Unit orimetric assay (Technicon Instruments, Ltd., and signed an informed consent. Exclusion crite- New York, N.Y.), while HDL cholesterol was de- ria included history of cardiovascular disease or termined enzymatically in the supernatant after stroke during the previous 36 months, total cho- precipitation of other lipoproteins with dextran lesterol >300 mg/dl, triglycerides >400 mg/dl, sulfate-magnesium. LDL cholesterol was calcu- blood pressure >140/90 mmHg, as well as the lated using Friedewald formula. use of sulphonylureas, thiazolidinediones, met- Haemoglobin A1c levels were measured by formin, insulin, glucocorticoids, antineoplastic using high-pressure liquid chromatography. Plas- agents, angiotensin receptor blockers, an- ma glucose levels were determined by using an giotensin converting enzyme inhibitors, psy- automated glucose oxidase method (Glucose choactive medications, drinking and/or smoking analyser 2, Beckman Instruments, Fullerton, habit. CA). Insulin was measured by RIA (RIA Diag- nostic Corporation, Los Angeles, CA) with a Procedure sensitivity of 0.5 mUI/L (normal range 0.5-30 All patients with a 2 weeks weight-stabiliza- mUI/L)15 and the homeostasis model assessment tion period before recruitment were enrolled. for insulin sensitivity (HOMA) was calculated Weight, blood pressure, basal glucose, c-reactive using these values16. CRP was measured by im- protein (CRP), insulin, insulin resistance munoturbimetry (Roche Diagnostcis GmbH, (HOMA), total cholesterol, LDL-cholesterol, Mannheim, Germany), with a normal range of HDL-cholesterol, triglycerides blood and (0-7 mg/dl) and analytical sensivity 0.5 mg/dl. adipocytokines (leptin, adiponectin, resistin, TNF-alpha, and interleukin 6) levels were mea- Adipocytokines sured at basal time. A tetrapolar bioimpedance, Resistin was measured by ELISA (Biovendor an indirect calorimetry and a prospective serial Laboratory, Inc., Brno, Czech Republic) with a assessment of nutritional intake with 3 days writ- sensitivity of 0.2 ng/ml with a normal range of ten food records were realized. Genotype of 4-12 ng/ml17. Leptin was measured by ELISA FABP2 gene polymorphism was studied. (Diagnostic Systems Laboratories, Inc., Min- neapolis, TX) with a sensitivity of 0.05 ng/ml Genotyping of FAPP2 Gene Polymorphism and a normal range of 10-100 ng/ml18. Oligonucleotide primers and probes were de- Adiponectin was measured by ELISA (R&D signed with the Beacon Designer 4.0 (Premier Systems, Inc., Minneapolis, MN) with a sensitiv- Biosoft International®, LA, CA). The polymerase ity of 0.246 ng/ml and a normal range of 8.65- chain reaction (PCR) was carried out with 50 ng 21.43 ng/ml19. Interleukin 6 and TNF alpha were of genomic DNA, 0.5 µL of each oligonucleotide measured by ELISA (R&D Systems, Inc., Min- primer (primer forward: 5’-CAG TTC CGT CTG neapolis, MN) with a sensitivity of 0.7 pg/ml and CTA GAT TGT-3’; primer reverse: 5’-GCT GAC 0.5 pg/ml, respectively. Normal values of IL6 AAT TAC ACA AGA AGG AA-3’), and 0.25 µL was (1.12-12.5 pg/ml) and TNFalpha (0.5-15.6 of each probes (wild probe: 5’-Fam-CAA AGA pg/ml)20-21.

90 ALA54THR polymorphism and diabetes mellitus type 2

Indirect Calorimetry test. Quantitative variables with normal distribu- For the measurement of resting energy expen- tion were analyzed with a two-tailed Student’s-t diture, subjects were admitted to a metabolic test. Non-parametric variables were analyzed ward. After a 12 h overnight fast, resting meta- with the U-Mann-Whitney test. Qualita tive vari- bolic rate was measured in the sitting awake sub- ables were analyzed with the chi-square test, ject in a temperature-controlled room over one with Yates correction as necessary, and Fisher’s 20 min period with an open-circuit indirect test. The statistical analysis was performed for calorimetry system (standardized for tempera- the combined Ala54/Thr54 and Thr54/Thr54 as a ture, pressure and moisture) fitted with a face group and wild type Ala54/Ala54 as second mask (MedGem; Health Tech, Golden, USA), group. Anova test was used to analized quantita- coefficient of variation 5%. Resting metabolic tive variables with three groups (Ala54/Thr54, rate (kcal/day) and oxygen consumption Thr54/Thr54 and Ala54/Ala54 patient groups). A (ml/min) were calculated22. p-value under 0.05 was considered statistically significant. Anthropometric Measurements Body weight was measured to an accuracy of 0.5 kg and BMI computed as body weight/(height2). Waist (narrowest diameter between xiphoid Results process and iliac crest) and hip (widest diameter over greater trochanters) circumferences to de- Fifty eight patients gave informed consent and rive waist-to hip ratio (WHR) were measured, were enrolled in the study. The mean age was too. Tetrapolar body electrical bioimpedance was 57.4±11.7 years and the mean BMI 37.8±6.5, used to determine body composition with an ac- with 15 males and 43 females. curacy of 5 g23. An electric current of 0.8 mA Thirty-three patients (56.9%) had genotype and 50 kHz was produced by a calibrated signal Ala54/Ala54 (wild type group) and 25 (43.1%) generator (Biodynamics Model 310e, Seattle, patients Ala54/Thr54 (19 patients, 32.8%) or WA) and applied to the skin using adhesive elec- Thr54/Thr54 (6 patients, 10.3%) (mutant group). trodes placed on right-side limbs. Resistance and Age was similar in both groups (wild type: reactance were used to calculate total body water, 57.3±11.7 yrs vs mutant group: 56.1±11 yrs: ns). fat and fat-free mass. Sex distribution was similar in both groups (wild Blood pressure was measured twice after a 10 vs mutant group), males (27.3% vs 24%) and fe- minutes rest with a random zero mercury sphyg- males (72.7% vs 76%). momanometer, and averaged. Table I shows anthropometric variables, with- out statistical differences. Table II shows cardio- Dietary Intake and Habits vascular risk factors. C-reactive protein, insulin Patients received prospective serial assessment and HOMA were higher in mutant group than of nutritional intake with 3 days written food wild group. records. All enrolled subjects received instruction Table III shows nutritional intake with 3 days to record their daily dietary intake for three days written food records. Calory, carbohydrate, fat, including a weekend day. Handling of the dietary and protein intakes were similar in both groups, data was by means of a personal computer without differences between allelic groups. equipped with personal software, incorporating Table IV shows levels of adipocytokines. No use of food scales and models to enhance portion differences were detected between genotypes. size accuracy. Records were reviewed by a regis- tered dietitian and analyzed with a computer- based data evaluation system. National composi- tion food tables were used as reference24. Discussion

Statistical Analysis The novel finding of this study is the associa- Sample size was calculated to detect differ- tion of the Thr54/Ala54 and Thr54/Thr54 FABP2 ences over 5% in insulin resistance with 90% genotypes with higher levels of insulin, HOMA power and 5% significance. The results were ex- and C reactive protein. pressed as mean ± SD. The distribution of vari- One hypothesis to explain these data has been ables was analyzed with Kolmogorov-Smirnov shown in a previous study. Baier et al.13 concluded

91 D.A. de Luis, M. Gonzalez Sagrado, R. Aller, O. Izaola, R. Conde, B. de La Fuente

Table I. Anthropometric variables.

Ala54/Ala54 (Ala54/Thr54 or Thr54/Thr54) Characteristics (n = 33) (n = 25)

BMI 37.4 ± 7.6 37.4 ± 5.3 Weight (kg) 94.7 ± 20.4 94.8 ± 15 Fat free mass (kg) 53.1 ± 14.9 51.2 ± 14.3 Fat mass (kg) 40.6 ± 17 43.6 ± 14.8 WC (cm) 112.1 ± 14 118.9 ± 13.7 Waist to hip ratio 0.96 ± 0.1 0.97 ± 0.09 Systolic BP (mmHg) 135.4 ± 21.1 138.5 ± 19 Diastolic BP (mmHg) 83.1 ± 9.1 81.4 ± 9.1 RMR (kcal/day) 1938 ± 421 2100 ± 805

RMR: resting metabolic rate. WC: Waist circumference. (*) p<0.05, in each group with basal values.

that threonine-containing protein may increase ab- inclusion criteria of subjects in previously studies sorption and/or processing dietary fatty acids by of the literature. For example, Carlsson et al31 the intestine and therefore increase fat oxidation, have detected higher concentrations of triglyc- which has been shown to inhibit glucose uptake in eride and cholesterol in the Thr54 allele patients, muscle and results in insulin resistance. in a population of obese patients with parenteral However, contradictory results have been de- history of cardiovascular disease. In a population scribed in the literature. Sipilainen et al.25 found of type 2 diabetes mellitus32, a linear relationship that obesity is not associated with specific vari- of mean fasting plasma triglyceride levels was ants in the FABP2 gene and that the Ala54-to- found and after fat ingestion, in homozygous for Thr polymorphism did not influence insulin lev- the Thr54 allele than in wiltdtype patients. In els in obese Finns. type 1 diabetes mellitus patients33 do not interact The frequency of the Thr54 alele variant is with the codon 54 polymorphism of the FABP2 43% in our study. Slightly lower frequency have gene to cause dyslipemia. Other population asso- been reported for other population as non diabet- ciation studies with insulin resistance and lipids ic Pima Indians (30%), Koreans (34%), Japanese in patients with type 2 diabetes mellitus34,35 were (35%), Swedish (30%) and white individuals esentially negative. Perhaps, the type of diabetes, from USA (32%)26-30. Perhaps, these different the time course of this disease, pharmacological frequencies and outcomes could be explained by treatments received by patients and comorbidities

Table II. Clasical cardiovascular risk factors.

Ala54/Ala54 (Ala54/Thr54 or Thr54/Thr54) Characteristics (n = 33) (n = 25)

Glucose (mg/dl) 132.8 ± 28 131.8 ± 22 Total ch. (mg/dl) 211 ± 38 220 ± 44 LDL-ch. (mg/dl) 138.4 ± 39 144.9 ± 36 HDL-ch. (mg/dl) 51.4 ± 10.5 52.7 ± 11.6 TG (mg/dl) 152.6 ± 58 163 ± 59 Insulin (mUI/L) 22.9 ± 11 26.6 ± 12* HOMA 7.65 ± 5.3 9.1 ± 6.3* CRP (mg/dl) 5.6 ± 4.2 8.7 ± 5.3* HbA1c (%) 5.8 ± 0.9 5.5 ± 0.7

Ch: Cholesterol. TG: Triglycerides CRP: c reactive protein. HOMA: Homeostasis model assessment. (*) p<0.05, in each group with basal values.

92 ALA54THR polymorphism and diabetes mellitus type 2

Table III. Dietary intake.

Ala54/Ala54 (Ala54/Thr54 or Thr54/Thr54) Characteristics (n = 33) (n = 25)

Energy (kcal/day) 1682 ± 555 1550 ± 497 CH (g/day) 171.5 ± 72 160.7 ± 66 Fat (g/day) 72.9 ± 30 66.7 ± 36 S-fat (g/day) 19.9 ± 11 17.1 ± 9.2 M-fat(g/day) 35.1 ± 14.3 30.9 ± 16.5 P-fat (g/day) 6.6 ± 3.2 5.6 ± 3.4 Protein (g/day) 82.4 ± 21 78.9 ± 21 Exercise (hs./week) 1.45 ± 2.8 1.38 ± 2.9 Dietary fiber 15.3 ± 6.1 14.7 ± 6.5

CH: Carbohydrate. S-fat: saturated fat. M-fat: monounsaturated fat. P-fat: polyunsaturated fat. No statistical differences.

could influence in these unclear previous results. In our study, the elevation of inflammation We propose studies with naïve diabetic patients markers (CRP) in patients with Thr allele, as as our design. shown in previous studies38 could be related with The lack of association with cholesterol and an altered postprandial response of fatty acids ab- triglyceride levels is clear in our study. Other Au- sorption (no measured in our design)39. Elevated thors have shown31,32 higher levels of cholesterol FFA increases the acummulation of triglycerides and triglycerides in non-diabetic obese patients in the adipocyte, related with imbalance of with Thr allele. Nevertheles, Duarte et al36 have lipoprotein lipase activity. The association of this shown a lower total and LDL cholesterol levels proimflammatory state with insulin resistance in in patients with Thr allele. These previous stud- Th carriers could indicate the existence of com- ies would have required composition analysis of plex gene-gene or gene-environment interactions the dietary intake to determine whether dietary that may enhance metabolic abnormalities in components could be responsible for the lipid these patients. profile modifications. For example, Marin et al37 In conclusion, the novel finding of this study is have shown that insulin sensitivity decreased in the association of the Thr54/Ala54 and subjects with Thr54 allele of the FABP2 poly- Thr54/Thr54 FABP2 genotypes with higher lev- morphism when saturated fatty acids were re- els of C reactive protein, insulin and HOMA. placed by monounsaturated fatty acids and car- Further studies are needed to elucidate the com- bohydrates. In our study dietary intake did not plex relationships40,41 among lipoprotein metabo- show statistical differences between groups, in lism, fat storage, inflammation markers and Thr this way our data have been controlled by dietary allele. A role of Ala54Thr polymorphism on inci- intake and previous discrepancies could be ex- dence of diabetes mellitus type 2 might be ex- plain by this uncontrolled factor (dietary intake). plored as a new association42.

Table IV. Circulating adypocitokines.

Ala54/Ala54 (Ala54/Thr54 or Thr54/Thr54) Characteristics (n = 33) (n = 25)

IL 6 (pg/ml) 3.34 ± 2.2 3.1 ± 2.5 TNF-alpha (pg/ml) 6.79 ± 3.8 6.4 ± 4.2 Adiponectin (ng/ml) 33.3 ± 30 28.5 ± 23 Resistin (ng/ml) 3.4 ± 1.8 3.7 ± 1.9 Leptin (ng/ml) 82.7 ± 66 108.4 ± 68

IL-6: interleukin 6. (*) p<0.05, in each group with basal values.

93 D.A. de Luis, M. Gonzalez Sagrado, R. Aller, O. Izaola, R. Conde, B. de La Fuente

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