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Original Studies |
Division of Endocrinology and Metabolic Diseases (R.L., M.E.B., G.S., A.M., G.C., A.L.), Laboratory of Clinical Chemistry (C.D.M.), Ospedale San Giuseppe, Istituto Auxologico Italiano, 28921 Verbania; and Molecular Biology Laboratory (E.P., A.M.D.B.), Istituto Auxologico Italiano Milano, 20135 Milano, Italy
Address correspondence and requests for reprints to: Anna Maria Di Blasio, M.D., Molecular Biology Laboratory, Istituto Auxologico Italiano, V.le Monte Nero 32, 20135 Milano, Italy. E-mail: biomol{at}auxologico.it
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Abstract |
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Introduction |
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Obese patients with relatively low leptin levels after correction for body mass index (BMI) have been described, and it has been hypothesized that this relative leptin deficiency could be responsible for the obesity of these patients (3). In addition, in Pima Indians, a population with marked genetic predisposition to obesity, relatively low plasma leptin concentrations precede weight gain (4). These findings prompted the investigators to hypothesize a role for regulatory regions of ob-gene expression. Quite recently, several studies reported the presence of different polymorphisms in the 5' noncoding region of the gene (5, 6, 7). Indeed, for one of these polymorphisms (A19->G), homozigosity for the G allele was found to be associated with leptin levels lower than those of patients with AG or AA genotype (6). However, these results were not confirmed by two other studies performed in a French and a Finnish population (5, 7). These contradictory data prompted us to investigate the presence of this polymorphism in a series of severe obese patients and to evaluate its possible influence on the variability of leptin levels and anthropometric, biochemical, and metabolic parameters related to leptin activity.
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Subjects and Methods |
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The present study included 205 obese patients (135 women and 70 men), 46.9 ± 14.23 yr old (mean ± SD; range, 18–81), with BMI values of 46.61 ± 6.68 kg/m2 (range, 36–85), referred to the Division of Endocrinology and Metabolic Diseases of the San Giuseppe Hospital, Istituto Auxologico Italiano (Piancavallo-Verbania) from September 1998 to May 1999, for diagnostic or therapeutic problems related to obesity or its morbidity.
Twenty-seven (16 women and 11 men) of the 205 patients had diabetes mellitus well controlled (glycated hemoglobin, 6.4 ± 1.5%) by diet alone. All patients had normal thyroid function, and none of them had concomitant severe renal, hepatic, or cardiac disease. Body weight was stable for the last 4 weeks before admission.
The patients underwent a study protocol including evaluation of BMI, WHR, serum leptin levels, resting energy expenditure (REE), energy intake, FM, total body water, total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides.
WHR was calculated on the basis of the measurements taken at patients’ admission; blood sampling to determine leptin levels and other biochemical parameters, assessment of REE and body composition were performed after a 12-h fast and before beginning any other treatment. Patients also underwent a 7-day dietary recall to estimate their usual daily energy intake.
The study protocol was approved by the Institution Ethics Committee; the aim and the design of the study were explained to the patients who gave their informed consent.
Genotype frequency of the patients was compared with that of 61 normal-weight (BMI, 21.05 ± 2.62 kg/m2) subjects (53 female and 8 male).
Measurement
Total body FM (kg), the percentage of fat body weight (FM), total body fat free mass (kg), and percentage of fat free mass were determined by the bioelectrical impedance analysis method (BIA 101/S Akern, Firenze, Italy) in the morning, after an overnight fast and after voiding.
REE was assessed by a computerized, open-circuit, indirect calorimetry system that measured resting oxygen uptake and resting carbon dioxide production using a ventilated canopy (Sensormedics, Milano, Italy). REE was measured at 0800 h, after an overnight fast, in a comfortable and thermo-regulated (22–24 C) room where only the investigator and the patient were present. After a 10-min steady-state period, values were recorded each minute for 30 min; the mean value was then expressed as kcal/24 h.
Body fat distribution was estimated using WHR. The waist circumference was taken to the smallest standing horizontal circumference between ribs and the top of the iliac crest, the hips circumference was taken as the largest standing horizontal circumference of the buttocks.
Glucose, total cholesterol, HDL cholesterol, and triglycerides were measured by enzymatic methods (Boehringer-Mannheim, Mannheim, Germany ); Hba1c values (Boehringer-Mannehim kits) were determined by immunoenzymatic methods (Tosoh, Kyobashi Chuo-Ku, Tokyo, Japan). FT4 and TSH were measured by RIA (DPC Euro/DPC Ltd, Llanberis, UK). Serum leptin levels were measured by RIA using reagents supplied by Linco Research, Inc. (St. Louis, MO). In this assay, the detection limit is 0.009 nmol/L; the intraassay variation is 2.2% at 0.375 nmol/L, 2.7% at 1.56 nmol/L, and 5.9% at 3.92 nmol/L; interassay variation from 10 different runs of 3 serum samples is 4.3%, 4%, and 6.9% at the concentration of 0.318, 1.31, and 3.5 nmol/mL respectively. In 32 lean subjects (BMI, 18–25 kg/m2), reference limits (2.5–97.5 percentiles) were 0.061–0.323 nmol/L in men and 0.162–1.08 nmol/L in women.
Polymorphism screening
The A19G variant present in the untranslated exon 1 of the ob gene was detected by PCR-restriction fragment length polymorphism, because the A->G substitution creates a restriction site for the Nsp BII enzyme.
One hundred nanograms of genomic DNA, isolated from whole blood, were amplified with the following primers: forward, 5'-CCCGCGAGGTGCACACTG-3'; and reverse, 5'-AGGAGGAAGGAGCGCGCC-3'.
PCR was carried out at 94 C for 5 min, followed by 30 cycles of 94 C for 20 sec, 58 C for 15 sec, 72 C for 20 sec, and a final extension step of 72 C for 5 min.
PCR products were visualized on a 2% agarose gel in the presence of ethidium bromide and purified by precipitation.
The products were incubated with 4 U of restriction endonuclease Nsp BII (Amersham Pharmacia Biotech, Little Chalfont, UK) in a total vol of 20 µL at 37 C overnight. The digestion product were analyzed on a 4% agarose gel, in the presence of ethidium bromide.
The wild-type allele was present as an undigested fragment of 221 bp; the mutant allele was present as two fragments of 183 bp and 38 bp.
Statistics
Unpaired t test was used to compare the clinical and demographic parameters between men and woman within obese subjects.
Contingency table chi-square tests were used to compare genotype frequencies between obese patients and control subjects.
The relationships between the A19G polymorphism and BMI, serum leptin concentration, and the other clinical parameters esteemed were studied by one-way ANOVA. Data are expressed as mean ± SD Energy intake was normalized for BMI; serum leptin levels were normalized for fat mass and for WHR; REE was normalized for BMI.
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Results |
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. All the
clinical parameters included, except BMI and serum total cholesterol,
are strongly associated with the gender. In particular, serum leptin
levels are significantly higher (P = 0.001) in women
than in men, also after adjustment for BMI and FM (data not shown).
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, in
our obese population, leptin levels, as well as the vast majority of
the other parameters tested, are not significantly different in
subjects homozygous for the G allele, when compared with subjects with
AG or AA genotypes. The only exception are obese women with the GG
genotype, who have a significantly lower WHR (P =
0.016).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Serum leptin concentration, particularly in obese patients, shows a high degree of variability. Both lean and obese women present leptin levels higher than those of men with similar BMI values (8, 9); the reasons for this sexual dimorphism are still unclear. Hormonal factors and gender differences in adipose tissue distribution have been considered, because sc adipose tissue, which is more active in synthesizing leptin than visceral one, is more represented in females than in males (10, 11). However, adiposity accounts for about 40% of the variability of leptin levels in obese patients (2), leaving a considerable proportion of this variability unexplained. For these reasons, the existence of genetic variants in the regulatory region of the ob gene has been extensively investigated. The presence of different polymorphisms in the 5' noncoding region of the gene has been recently reported (5), but contradictory results have been obtained on the association between these polymorphisms and variations of leptin levels in obese subjects. The C-2548->A substitution was associated with higher leptin levels (5), whereas patients bearing the A19->G polymorphism showed leptin levels either no different (5, 7) or lower (6), when compared to those of obese patients with comparable values of BMI but without this polymorphism.
Because of these different data reported, we decided to further investigate the importance of the genetic component as one of the factors involved in the variability in leptin levels, and thus, we looked to the A19->G substitution in the Italian population. In agreement with the data by Mammes et al. (5) and by Karvonen et al. (7), we also did not find any association between decreased leptin levels and the presence of the G allele. This is in contrast with the study of Hager et al. (6). Indeed, these authors found lower leptin levels in obese patients homozygous for the G allele, in absence of any difference in fasting glucose and insulin levels and BMI values. However, it must be pointed out that, in their study, WHR values were not reported; thus, it is not possible to rule out that the results obtained were attributable to different fat distribution.
In the present study, we investigated a severe obese population (BMI values always higher than 40). Moreover, these subjects were also well characterized because, beside leptin, many other obesity-related metabolic characteristics were taken into account. Because of known gender differences, the possible association between genotype and clinical parameters was investigated separately for men and women. In both groups, homozygosity for the G allele was not associated with significant differences in the variables considered, with the exception of WHR values, which were significantly lower in women. At present, the physiological relevance of this finding remains unclear.
In conclusion, the present study, performed on a large Italian obese population, does not suggest, in agreement with what already has been reported by other investigators (5, 7), a significant role for the A19->G polymorphism in determining leptin values.
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Footnotes |
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Received January 3, 2000.
Revised July 3, 2000.
Accepted July 7, 2000.
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References |
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  V. Paracchini, P. Pedotti, and E. Taioli Genetics of Leptin and Obesity: A HuGE Review Am. J. Epidemiol., July 15, 2005; 162(2): 101 - 114. [Abstract] [Full Text] [PDF]
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