Leptin (gr. Leptos – lean) is 16 kDa polipeptide hormone and a product of the obesity gene (ob gene), first described by Zhang in 1994 (1). This hormone is secreted mostly by adipose tissue. Leptin acts as a satiety factor – by inhibiting neuropeptide Y in the hypothalamus, provides a satiety signal with subsequent increase of energy expenditure and metabolic processes intensification. It also regulates the amount of fat tissue in organism, takes part in fertility processes and regulates blood pressure (2-4). The leptin gene is located on the long arm of chromosome 7 (7q31.3) and contains 3 exons and 2 introns. Several polymorphisms of the leptin gene were described, such as functional polymorphism V110M, promoting region polymorphism -188 C/A and -2548 G/A. Many authors have presented research which indicates that differences in the leptin genotype are associated with overweight individuals (5-8). Leptin acts via specific receptors located in adipose tissue, stomach, endometrium, liver, spleen, lungs, heart, ovaries and placenta. A high amountof leptin receptor was discovered in the hypothalamus, where leptin acts via negative loop feedback between its concentration in blood serum and its receptor expression on the cell surface (9-11). There are several isoforms of leptin receptor, known as long and short isoforms. In humans four isoforms were identified: 1165 amino-acid long isoform, responsible for leptin signaling and 3 alternative splicing short isoforms (12-14). The leptin receptor is a translation product of the leptin receptor gene (LEPR gene) located on chromosome 1 (1p31) which contains 20 exons (15). Both the leptin gene and its receptor gene are highly polymorphic. Several genotypic variants of investigated genes may affect leptin blood concentration and the biologic function of its receptor, thus influencing body weight. The most frequent leptin gene receptor polymorphisms are: functional -668 A/G and -109 A/G in exon 6, silent mutation -343 T/C and -1019 G/A. Pregnancy and diabetes are especially associated with body mass fluctuations.

The aim of the study was to investigate the frequency of occurrence of both the leptin gene and its receptor polymorphisms in pregnant women with type 1 diabetes mellitus (PGDM-1) and to estimate the association of several genotypes in normal weight and over-weight subjects. Moreover we aimed to evaluate the frequency of alleles in studied groups. Additionally, the relationship between metabolic parameters describing glycemic control and genotypes were evaluated.


MATERIAL AND METHODS
78 Caucasian pregnant women having a single pregnancy with type 1 diabetes mellitus were qualified to the study group. All women were hospitalized in the Department of Obstetrics and Women Diseases in Poznañ Poland. The study group was divided into 4 subgroups depending on the BMI value (16). The first subgroup consisted of 3 underweight subjects (body mass index- BMI 18,5 kg/m2), second of 35 normal weight subjects with BMI between 18,5- 24,9 kg/m2, the third subgroup consisted of 27 subjects with BMI between 25 - 29,9 kg/m2 and fourth subgroup consisted of 13 subjects with BMI 30 kg/m2. The control group consisted of 34 healthy Caucasian women with a single pregnancies and with normal body weights (BMI 24, 9 kg/m2).

Serum concentration of the following metabolic parameters were measured: MBG-mean blood glucose (mmol/l), HbA1C (%), LDL-cholesterol (mmol/l), HDL-cholesterol (mmol/l), total cholesterol (mmol/l), insulin dosage (Units), creatinin level (µmol/l), creatinin clearance (ml/min). The glucose level in serum of venous blood was determined by means of the enzymatic (heksokinase) method with the Roche Diagnostics laboratory reagents on Hitachi 912 analyzer.

The percentage of glycated hemoglobin (HbA1c) in capillary blood was estimated using the Roche Diagnostics Tina-quant® Hemoglobin A1C II test.

The total serum cholesterol, HDL cholesterol and triglycerides levels were measured with the appropriate Roche Diagnostics reagents (Cholesterol CHOD-PAP, HDL-C plus and Triglycerides GPO-PAP respectively) on Hitachi 912 analyzer, and LDL cholesterol level was calculated using the formula: LDL cholesterol = total cholesterol – HDL cholesterol – TG/5.

The daily urine protein loss (g/24h), serum creatinine level (µmol/l), creatinine clearance (ml/min) were estimated by using Jaffe modified test.

Genetic analysis

In all subjects (SG and CG), the leptin gene and leptin gene receptor polymorphisms (LEP 2548 G/A and LEPR 668 A/G respectively) were evaluated by Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) assays. DNA was extracted from leucocytes using QIAamp DNA Blood mini KIT (Qiagen Inc. Germany).

PCR was performed for genotype analysis using PTC 100 Programmable Thermal Controller (MJ Research INC USA.). The following substrates were added: DNA sample, distilled water, (NH4)2SO4 buffer, MgCl2, dNTPs, primers, Taq DNA polymerase. PCR was performed in three stages: high temperature DNA denaturation (94°C), primers adding after temperature drop, essential replication (35 PCR cycles). The PCR product after digestion was transferred on 1,5% agarose gel (80V, 120 min) with TBE buffer (Sigma) and visualized in ultraviolet light.

As a next step, the PCR product was digested with specific restriction enzymes: Cfo1 5’...GC(G/A)CT…3’ (Fermentas, Litwa) to identify genotypic variants within promoting region polymorphism of the leptin gene and MspI 5’…C/CGG…3’ (EURx, Poland) to estimate polymorphisms of the leptin gene receptor. All procedures were performed according to the following manufacturer recommendations: digestion temperature 37°C (16 hours), and enzyme inactivation temperature 65°C (20 min.). The following genotypes were found, including -2548 G/A: heterozygote GA 242bp, 181bp, 61bp, homozygote GG 181bp, 61bp, mutated homozygote AA 242bp. Including LEPR 668 A/G: heterozygote AG 216pz, 134pz, 82pz, homozygote AA 216bp, mutated homozygote GG 134bp, 82bp. All molecular biology investigations were performed in the Molecular Biology Laboratory in Department of Perinatology and Women Diseases, Karol Marcinkowski University of Medical Sciences.

Moreover the patients’ age, height (cm), first trimester weight (kg), systolic and diastolic blood pressure (mmHg) were determined.

Statistical analysis

The frequency of observed genotypes and alleles are shown as a percentage of the whole group whereas expected values were estimated according to Hardy-Weinberg equilibrium. The statistical significance between genotypes in the study subgroups were estimated using the Chi-squared test. Differences between biochemical parameters in relation to genotypes were estimated by one-way Anova and the post-hoc tests. Differences were acknowledged as statistically significant with p<0, 05. SPSS 16.0 for Windows was used for statistical analyses. To conduct the study, approval was granted by the Local Ethics Committee of the University.


RESULTS
Figs 1-5 describes the frequency of observed genotypes and alleles of the leptin gene polymorphism (-2548 G/A) in the study subgroups and the control group in relation to BMI. With the increasing value of BMI in the study subgroups, there was a trend observed of G and A homozygous genotypes among the majority of SG subjects, however it did not reach statistical significance probably due to the low number of under- and over-weight subjects. In the control group which consisted of healthy lean individuals, a majority of GA heterozygous genotypes were observed, which corresponds to the frequency of heterozygous variants in normal weight, and diabetic subjects. There was a trend observed in the studied subgroups of a majority having allel A. Similar results were observed in relation to the frequency of occurrences of the leptin gene receptor polymorphism (668 A/G). With the increasing value of BMI in the first trimester, there was a trend to G and A homozygous majority genotypes noticed, however without statistical significance. In the control group, a majority of GA heterozygous genotypes were observed, which also corresponded to the frequency of this variant in the lean diabetic women. The above results are shown in Figs 6-10.

Figs 1AB-5AB. Frequency of LEP 2548 G/A and its alleles in the study and control group in relation to BMI in first trimester of pregnancy (Chi-squared test).

In the study group we analyzed the relationship between the observed genotypes and some biochemical parameters describing lipid and glycemic controls. We did not noticed statistical significant differences in the parameters in relation to observed genotypes of the leptin gene polymorphism and leptin gene receptor polymorphism. The relationships between these parameters are shown in Table 1 and 2.

Figs 6AB-10AB. Frequency of LEPR 608 A/G and its alleles in the study and control group in relation to BMI in first trimester of pregnancy (Chi-squared test).

Since the majority of homozygous genotypes were found in the group of patients who were noted to be overweight and obesity, we evaluated the relationship between biochemical parameters in these groups. We found statistically significant differences in concentrations of triglycerides, HDL-cholesterol and creatinine clearance between analyzed subgroups of the diabetic pregnant women (Table 3).

Table 1. Metabolic parameters and observed genotypes in leptin gene (LEP 2548 G/A) in first trimester pregnant women with type 1 diabetes mellitus.

*-One-way Anova, post-hoc tests – Levene, Scheffe, Tukey

Table 2. Metabolic parameters and observed genotypes in leptin gene receptor ( LEPR 668 A/G) in first trimester pregnant women with type 1 diabetes mellitus.

*-One-way Anova, post-hoc tests – Levene, Scheffe, Tukey

Table 3. Metabolic parameters in the study group according to patients’ BMI value

*-One-way Anova, post-hoc tests – Levene, Scheffe, Tukey

We also studied whether there was any difference in the presence of studied metabolic parameters and the combination of possible genotypes including both the leptin gene and the leptin gene receptor polymorphisms. Since there are 3 possible genotypes including LEP -2548 G/A (GA, GG and AA) and 3 possible genotypes including LEPR 668 A/G (AG, AA, GG), we established 9 groups of patients in the diabetic group (e.g. LEP GA heterozygote and LEPR AA homozygote or LEP GA heterozygote and LEPR GG homozygote etc.), depending on genetic configuration. The same was performed in the control group. In the study group, no specific genotype was significantly related to obesity. We also did not find statistically significant differences in any of the studied biochemical parameters between these subgroups. In the control group no specific genotype was dominantly observed.


DISCUSSION
Mutations in the leptin gene and the leptin gene receptor may lead to extreme obesity, this was found to be true in mice by Chen et al. (17), and in humans by Clement et al. (18). They found, that the presence of a homozygous mutations in the leptin gene (homozygotes ob/ob) and also homozygous mutations in the leptin gene receptor (homozygotes db/db) were associated with early onset of extreme obesity due to hyperphagia, poor energy expenditure and severe insulin-resistance (17, 18). These studies gave rise to the idea that several genotypes of the leptin gene and its receptor may lead to obesity.

The leptin gene and the leptin gene receptor are polymorphic, which means that in different subjects, differences in genetic variants may be found. Investigations involving these polymorphisms were conducted in relations to obesity in different populations. Wang et al. (5) performed a study on 200 Taiwanese subjects with extreme obesity, where the relationship between obesity and several polymorphism among the promoting region of the leptin gene -2548 G/A and the leptin gene receptor 668 A/G was analyzed. They found, that the homozygous variants of the LEP -2548 G/G were strongly associated with the development of extreme obesity, whereas no specific association between obesity and the leptin gene receptor polymorphism was discovered (5).

Several studies conducted in Caucasian and Afro-American populations discovered that common leptin gene polymorphisms in the flanking region -2548 G/A may affect the level of circulating leptin in humans, which was also associated with an increase in birth weight that depending on gender. It was found that allele A was related to an increase in female size for gestational age, while the G allele was associated with decreased male birth in the Caucasian population. Among African-Americans, the A allele was associated with a decrease in the umbilical cord leptin in females and with an increase in the cord leptin in males (19).

Obesity is the result of an imbalance between food intake and energy expenditure resulting in the storing of energy as fat, in most cases due to specific eating patterns. The study conducted by de Krom et al. (20) on a large study population was focused on identifying whether carriers of a common leptin receptor and cholecystokinine gene polymorphisms are genetically predisposed to obesity. It was found that common genetic variations of the leptin gene receptor are related to specific snacking behavior, whereas the carriers of cholecystokinine polymorphisms had an increased risk of eating enlarged meal sizes. Also in the Australian population of obese women, Pro1019Pro leptin gene receptor polymorphism was associated with longitudinal increases in body weight, fat mass and body mass index. It was also discovered that individuals homozygous for the A allele at this locus had a greater susceptibility to gain body weight. No association was found in relation to variation in the beta-3 adrenergic receptor Trp64Arg, tumor necrosis factor-alpha promoter, or leptin genes in non-obese or obese women. This conclusion confirms the hypothesis that in most of the cases the leptin gene receptor can be involved in the development of obesity (21). In the Spanish population, the leptin gene receptor variants (Q223R) can also be associated with obesity, rather than a -2548 G/A leptin gene polymorphism (6). Similar results were obtained for Brazilian population (7)

Studies including lipid profile were carried in normal and over-weight populations. Van der Vleuten et al. discovered the presence of homozygous genotypes rather than heterozygous for the leptin gene receptor may be associated with familiar combined hyperlipidemia (22).

In our study, we found that with the increasing value of BMI in first trimester of pregnancy complicated by diabetes mellitus type 1, increased the tendency for the presence of homozygous A and G genotypes in both the leptin gene and the leptin gene receptor. It is worth mentioning that it may be necessary to recruit more patients into study, especially those who were noted to be under and overweight in order to reinforce our findings. Since our results did not reach statistical significance, we may be able to conclude that specific trends were discovered. Our data correspond with results reached by other investigators as mentioned above. In the group of lean diabetic women in comparison to lean healthy subjects, no specific differences in the frequency of observed genotypic variants were noted. In both groups a majority of heterozygous variants were found, which may suggest that obesity and being overweight may be associated with homozygous variants of the leptin gene and its receptor.

We also tried to find association between investigated polymorphisms and biochemical parameters describing glycemic control, lipid profile, the blood pressure value and other parameters presented in table 1 and 2. We observed that in first trimester of pregnancy there are no specific or statistically significant differences in the studied parameters in these groups of women, however when analyzing the whole groups of patients with different BMI, where the trend to homozygous variant was discovered, the statistical differences among lipid parameters were found.

In world-wide literature, there is only one study describing the influence of the leptin gene receptor polymorphism (Q223R, 668 A/G) on body weight gain during first trimester pregnancy, the levels of circulating leptin and fetal body weight. This study performed was performed on on 455 women of different races having healthy pregnancies and showed no significant association with maternal BMI in early pregnancy and with fetal body weight (23). There are however some studies describing the role of leptin and changes of its concentrations and leptin polymorphisms during normal pregnancy and complicated by pre-eclampsia and gestational diabetes (24-26). There are no studies describing the possible role of the selected leptin gene and its receptor polymorphisms on body weight gain during pregnancy complicated by diabetes mellitus type 1. Thus our study allows us the possibility to monitor this group of diabetic women during pregnancy, and their weight gain changes, and correlate them with defined leptin polymorphisms as well as with changes of serum leptin concentrations that will be monitored in relation to the studied polymorphisms. The possible role of those factors will also be analyzed.

Since a trend to an increased number of homozygotic variants in both the leptin gene and the leptin gene receptor in over-weight subjects were found, it is necessary to include more patients to the study to confirm this result. It is also necessary to conduct the study in the same groups of patients during the whole term of the pregnancy, to establish if genetic variants of the investigated polymorphisms are associated with elevated body weight in the second and third trimester. This will also let us confirm the association between genetic variants and glycemic controls, lipid control, and the development of specific diabetic complications during pregnancy. It has also been found that several endocrine complications related to pregnancy are associated with multi-genetic factors, therefore, in every subject, it is necessary, to take under consideration simultaneously both investigated genes and its polymorphisms as risk factors for the development of these complications and to avoid considering it separately.

Acknowledgements: The study was supported by the grant N N407 2783 33.

Conflict of interest statement: None declared.


REFERENCES

1. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedmand JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994; 372: 425–432.
2. Montague CT, Farooqi IS, Whitehead JP et al. Congenital leptin deficiency is associated with severe early-onset obesity in humans. Nature 1997; 387: 903-8.
3. Sinha MK. Human leptin: the hormone of adipose tissue. Eur J Endocrinol 1997; 136: 461–464.
4. Zwirska-Korczala K, Adamczyk-Sowa M, Sowa P, Pilc K, Suchanek R, Pierzchala K, Namyslowski G, Misiolek M, Sodowski K, Kato I, Kuwahara A, Zabielski R. Role of leptin, ghrelin, angiotensin II and orexins in 3T3 L1 preadipocyte cells proliferation and oxidative metabolism. J Physiol Pharmacol. 2007; 58(Suppl 1): 53-64.
5. Wang TN, Huang MC, Chang WT et al. G-2548A polymorphism of the leptin gene is correlated with extreme obesity in Taiwanese aborigines. Obesity 2006; 14: 183-187.
6. Portolés O, Sorlí JV, Francés F et al. Effect of genetic variation in the leptin gene promoter and the leptin receptor gene on obesity risk in a population-based case-control study in Spain. Eur J Epidemiol 2006; 21: 605-612.
7. Duarte SF, Francischetti EA, Genelhu-Abreu V et al. Q223R leptin receptor polymorphism associated with obesity in Brazilian multiethnic subjects. Am J Hum Biol 2006; 18: 448-453.
8. Popko K, Gorska E, Wasik M et al..Frequency of distribution of leptin receptor gene polymorphism in obstructive sleep apnea patients. J Physiol Pharmacol 2007; 58(Suppl 5/Pt 2): 551-561.
9. Couce ME, Burquera B, Paresi JE, Jensen MD, Lloyd RV. Localization of leptin receptor in the human brain. Neuroendocrinology 1997; 66: 145–150.
10. Weigle DS. Leptin and other secretory products of adipocytes modulate multiple physiological functions. Ann Endocrinol 1997; 58: 132– 136.
11. Smolinska N, Siawrys G, Kaminski T, Przala J. Leptin gene and protein expression in the trophoblast and uterine tissues during early pregnancy and the oestrous cycle of pigs. J Physiol Pharmacol 2007; 58(3): 563-581.
12. Tsiotra PC, Pappa V, Raptis SA, Tsigos C. Expression of the long and short leptin receptor isoforms in peripheral blood mononuclear cells: implications for leptin’s actions. Metabolism 2000; 49: 1537-1541.
13. Papadopoulos DP, Makris TK, Poulakou M et al. Human soluble leptin receptor concentration in healthy offspring of hypertensive parents. J Clin Hypertens 2006; 8: 797-802.
14. Siawrys G, Kaminski T, Smolinska N, Przala J. Expression of leptin and long form of leptin receptor genes and proteins in pituitary of cyclic and pregnant pigs. J Physiol Pharmacol 2007; 58(4): 845-857.
15. Thompson DB, Ravussin E, Bennett PH et al. Structure and sequence variation at the human leptin receptor gene in lean and obese Pima Indians. Hum Mol Genet 1997; 6: 675–679.
16. Kinalska I, Telejko B, Kinalski M. Oty³oœæ i cukrzyca. In: Cukrzyca, J. Sieradzki (red). Gdañsk, Via Medica 2006, pp. 282- 311.
17. Chen H, Charlat O, Tartaglia LA et al. Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell 1996; 84: 491–495.
18. Clement K, Vaisse C, Lahlou N et al. A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature 1998; 392: 398–401.
19. Adkins RM, Klauser CK, Magann EF et al. Site -2548 of the leptin gene is associated with gender-specific trends in newborn size and cord leptin levels. Int J Pediatr Obes 2007: 16: 1-8.
20. de Krom M, van der Schouw YT, Hendriks J et al. Common genetic variations in CCK, leptin, and leptin receptor genes are associated with specific human eating patterns. Diabetes 2007; 56: 276-280.
21. de Silva AM, Walder KR, Boyko EJ et al. Genetic variation and obesity in Australian women: a prospective study. Obes Res 2001; 9: 733-740.
22. van der Vleuten GM, Kluijtmans LA, Hijmans A, Blom HJ, Stalenhoef AF, de Graaf J. The Gln223Arg polymorphism in the leptin receptor is associated with familial combined hyperlipidemia. Int J Obes 2006; 30: 892-898.
23. Rand L. Winchester EC, Milwood IY, Penny MA, Kessling AM. Maternal leptin receptor gene variant Gln223Arg is not associated with variation in birth weight or maternal body mass index in UK and South Asian populations. Int J Obes Relat Metab Disord 2001; 25: 753-755.
24. Rigo J, Szendei G, Rosta K et al. Leptin receptor gene polymorphisms in severely pre-eclamptic women. Gynecol Endocrinol 2006; 22: 521-525.
25. Martinez-Abundis E, Gonzalez-Ortiz M, Pascoe-Gonzalez S. Serum leptin levels and the severity of preeclampsia. Arch Gynecol Obstet 2000; 264: 71–73.
26. Stock SM, Bremme KA. Elevation of plasma leptin levels during pregnancy in normal and diabetic women. Metabolism 1998; 47: 840–843.