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The Journal of Maternal-Fetal and Neonatal Medicine, 2013; 26(2): 123–126© 2013 Informa UK, Ltd.ISSN 1476-7058 print/ISSN 1476-4954 onlineDOI: 10.3109/14767058.2012.728648

Objective: To investigate possible alterations in cord blood levels of adipokines, chemerin and obestatin (secreted by adipose tissue and associated with later development of insulin resistance/metabolic syndrome), as well as insulin, in large for gestational age (LGA) and appropriate for gestational age (AGA) pregnancies, granted that these groups differ in body fat mass and metabolic/endocrine mechanisms. Methods: Cord blood chemerin, obestatin, and insulin concentrations were prospec-tively measured in 40 LGA (9 born from diabetic and 31 from nondiabetic mothers) and 40 AGA singleton full-term infants. Results: Cord blood chemerin concentrations were significantly higher in LGA compared with AGA neonates (b = 38.91, SE 9.29, p < 0.001). In contrast, no significant differences in obestatin concentrations were observed between groups. Insulin levels were significantly elevated as customized centiles increased (b = 0.003, SE = 0.001, p = 0.032). Conclusions: Higher chemerin concentrations in LGA neonates possibly reflect the increased adipose tissue in this group. Lack of difference between the two groups in circulating levels of obestatin–possibly a sensitive marker of insulin resistance–might be due to development of metabolic disorders later in life.

Keywords: Adipokines, fetus, insulin resistance, neonate

Abbreviations: AGA: appropriate for gestational age; GDM: gestational diabetes mellitus; LGA: large for gestational age; T2DM: type 2 diabetes mellitus

IntroductionThe pattern of intrauterine growth has been shown to be asso-ciated with disturbances in glucose metabolism in later life [1]. Children born large for gestational age (LGA) have an increased risk of developing insulin resistance, metabolic syndrome, diabetes, and early cardiovascular disease [2].

Insulin is an important regulator of fetal growth. Prolonged hyperinsulinemia is accompanied by fat deposition in the fetus and, thus, by increased production of adipose tissue resulting in fetal overweight [3]. Adipose tissue has been recognized as an active endocrine organ that regulates body energy homeostasis, through the production of several adipokines [4,5]. Obesity is linked to the metabolic syndrome, by an altered adipokine profile, insulin resis-tance, obesity-related diabetes, and relative metabolic disorders [6].

Chemerin, also known as tazarotene-induced gene 2 [7], has been recently characterized as a chemotactic agent that was iden-tified as the ligand for an orphan G protein-coupled receptor, ChemR23 expressed by immature dendritic cells and macro-phages [8]. Chemerin is a novel adipokine of 16 kD molecular weight [9], implicated in adipogenesis and adipocyte metabolism [10]. Chemerin mRNA is highly expressed in mature adipocytes and found increased in adipose tissue of obese animals [11]. Furthermore, chemerin enhances insulin-dependent glucose uptake in adipocytes [12], thus, suggesting the pathophysi-ological significance of chemerin in obesity and diabetes. There is not definite evidence about the source of chemerin in cord blood, however, human placenta is shown to express chemerin mRNA, whereas chemerin was found by immunohistochemistry in placental cells [9]. On the other hand, a transplacental transfer of chemerin is not established; nevertheless, its relatively high molecular weight might prevent it [9].

Obestatin, a 23-amino acid peptide, is a novel hormone that is encoded by the ghrelin gene and is generated from proghrelin by cleavage with a convertase [13]. Ghrelin and obestatin, although coded by the same gene, are claimed to exercise opposite physi-ological effects [14]. Obestatin plasma levels were found to be lower in obese subjects compared with women of normal weight and anorexic patients [15]. In addition, decreased obestatin levels were found in patients with type 2 diabetes mellitus (T2DM) [13], suggesting that obestatin production is linked to adiposity and insulin resistance [16].

In this study, we hypothesized that circulating chemerin, obestatin, and insulin levels should differ between LGA cases and appropriate for gestational age (AGA) controls, since the former present with increased fat mass and undergo adaptational changes of endocrine/metabolic mechanisms, due to excessive intrauterine growth, possibly leading to insulin resistance. More specifically, we hypothesized that chemerin levels should be increased and obestatin levels decreased in LGA as compared with AGA infants. Therefore, we aimed to evaluate and correlate circulating chemerin, obestatin, and insulin concentrations in umbilical cord blood of LGA and AGA neonates.

MethodsThe Ethics Committee of our teaching hospital approved the study protocol and all parturients provided signed informed consent

Cord blood chemerin and obestatin levels in large for gestational age infants

Theodora Boutsikou1, Despina D. Briana1, Maria Boutsikou1, George Kafalidis1, Lamprini Stamati2, Stavroula Baka1, Demetrios Hassiakos1, Demetrios Gourgiotis2 & Ariadne Malamitsi-Puchner1

1Neonatal Division, 2nd Department of Obstetrics and Gynecology, Athens University, Medical School, Athens, Greece and 2Research Laboratory of Clinical Biochemistry-Molecular Diagnostics, 2nd Department of Pediatrics, Athens University, Medical School, Athens, Greece

Correspondence: Ariadne Malamitsi-Puchner, MD, 19, Soultani Street, 10682 Athens, Greece. Tel: +30 6944443815; Fax: + 30 2107233330. E-mail: amalpu@aretaieio.uoa.gr

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before recruitment. Forty AGA and 40 LGA infants were included in the study. As AGA were considered infants with a birth weight between the 10th and 90th customized centile, whereas LGA were infants with a birth weight above the 90th customized centile and above 4000 g in weight. The Gestation-Related Optimal Weight (GROW) computer-generated programme [17,18] was used to calculate the customized centile for each pregnancy. Significant determinants of birth weight, such as maternal height and booking weight, ethnic group, parity, gestational age, and sex, were entered into the program to adjust the normal birth weight centile limits [17].

Gestational diabetes mellitus (GDM) was identified as the cause of LGA in 9 out of the 40 pregnancies.

All neonates presented no symptoms of intrauterine infection or signs of genetic syndromes; 1- and 5-min Apgar scores were ≥8 in all LGA cases and AGA controls.

The demographic data of participating mothers and infants are listed in Table I.

Mixed arteriovenous blood was collected from the doubly clamped umbilical cord reflecting fetal state. Blood was collected in pyrogen-free tubes and was immediately centri-fuged after clotting. The supernatant serum was kept frozen at −80 °C until assay. The determination of chemerin levels was performed in the total 80 blood samples by Enzyme-Linked Immunosorbent Assay (Biovendor 621 00 Brno, Chez Republic). The minimum detectable concentration, intra- and interassay coefficients of variation were 0.1 ng/ml, 7 and 7.5%, respectively.

The determination of obestatin levels was performed in the total 80 blood samples by Enzyme-Linked Immunosorbent Assay (Biovendor 621 00 Brno, Chech Republic). The minimum detect-able concentration, intra- and interassay coefficients of variation were 0.231 ng/ml, 6 and 7.5%, respectively.

In addition, serum insulin levels in all 80 samples were evalu-ated by Immuno Radiometric Assay (Immunotech s.r.o, 10227 Prague, Czez Republic). The minimum detectable concentration, intra- and interassay coefficients of variation were 0.5 μU/ml, 3.4 and 4.3%, respectively.

Statistical analysis

Chemerin data were normally distributed (Kolmogorov-Smirnov test); thus, parametric tests were applied in the statistical analysis. Linear regression analysis was used to examine the effects of various parameters (maternal age, centile, birth weight, mode of delivery, gestational age, gender, parity, GDM) on circulating chemerin concentrations.

Obestatin data did not follow normal distribution, even after logarithmic transformation, thus nonparametric procedures were applied in the analysis. Mann-Whitney U-test was used to detect differences in obestatin levels in regard to group, gender, type of delivery, parity, and GDM. Pearson’s or Spearman’s correlation coefficient was used, where appropriate, to examine any posi-tive or negative correlations between obestatin and birth weight, centile, gestational age, and maternal age.

Variables regarding insulin did not follow normal distribution, thus, logarithmic transformation was applied. Linear regression analysis was used to estimate the effect of different factors (group, gender, birth weight, gestational age, mode of delivery, centile, maternal age, parity, and GDM) on insulin levels. Student’s t-test or Mann-Whitney U-test was used, where appropriate, to detect differences in quantitative variables between the two groups (LGA − AGA). Pearson’s χ2 test was used to estimate differences between categorical variables. p <0.05 was considered statistically significant.

ResultsDetermined values of circulating chemerin, obestatin, and insulin levels in both groups are shown in Figures 1–3. Umbilical cord chemerin levels were significantly elevated in the LGA compared with the AGA group (b = 38.91, SE 9.29, p < 0.001).

No significant differences were demonstrated in obestatin levels between LGA and AGA fetuses. Obestatin levels did not present any significant alterations in respect to group, gender, mode of delivery, parity, and GDM. No significant correlations between obestatin and chemerin were observed.

Table I. Demographic data of participating mothers and their appropriate for gestational age (AGA) and large for gestational age (LGA) fetuses-neonates.

VariablesAGA (Mean ± SD/

median (range))LGA (Mean ± SD/median (range)) p value

Birth weight (g) 3318.75 ± 234.14 4248.5 ± 228.93 <0.001Gestational age (weeks) 39.27 ± 1.0 39.26 ± 1.02 NSCentile 42.5 (12.0–72.0) 97.0 (90–100) <0.001Maternal age (years) 32.5 ± 3.88 30.70 ± 4.63 NSGender, N (%) NS Male 20 (50) 27(67.5) Female 20 (50) 13(32.5)Mode of delivery, N (%) NS Vaginal 18 (45) 18 (45) Cesarean section 22 (55) 22 (55)Parity, N (%) NS First 23 (57.5) 23 (57.5) Other 17 (42.5) 17 (42.5)Gestational diabetes mellitus, N (%)

0.002

No 40 (100) 31 (77.5) Yes 0 (0) 9 (22.5)AGA, appropriate for gestational age; LGA, large for gestational age; NS, not significant.

Figure 1. Chemerin levels in umbilical cord serum of appropriate for gestational age (AGA) and large for gestational age (LGA) fetuses. Error bars represent mean (95% CI). CI, confidence interval.

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The effect of customized centile on insulin cord blood levels was proved significant, thus, insulin levels were significantly elevated as customized centile increased (b = 0.003, SE 0.001, p = 0.032).

Finally, no significant correlations were observed between obestatin/chemerin and insulin serum concentrations in either group.

DiscussionIt is well established that obese individuals who suffer from the metabolic syndrome display a characteristic imbalance of their adipokine profile, which leads to severe changes in insulin

sensitivity and finally increased occurrence of metabolic disor-ders [6]. The imbalance and dysregulation of these adipokines usually draw their origin to the fetal development in utero, where nutrient availability to the fetus might define either the excessive or diminished adipose tissue formation [19,20]. Furthermore, pregnancy enhances an altered adipokine profile, due to changes in body mass index and adipokine synthesis in the placenta [21].

Insulin is a potent regulator of fetal growth. High fetal insulin levels lead to increased fat deposition in the fetus and thus, accu-mulation of adipose tissue, resulting in excessive birth weight [3]. In this respect, this study has shown the statistically significant elevation of insulin cord blood levels with increasing customized centile.

As mentioned above, LGA infants have an enhanced risk of developing insulin resistance and metabolic syndrome later in life [2]. Maternal obesity, excessive weight gain during pregnancy, and GDM are the main causes of LGA. However, there is a large group of LGA pregnancies which present none of the above risk factors [22]. It has been suggested that in these cases the cause of LGA is unidentified hyperglycemia during pregnancy, resulting in hyperinsulinemia in utero and leading to the aforementioned metabolic adaptations later in life [23]. The above might explain why the LGA population in this study presents altogether with an altered metabolic profile (hyperinsulinemia, increased chemerin levels), although only in a small proportion of the sample the cause of LGA is GDM.

In addition, our results showed umbilical cord chemerin levels to be significantly elevated in the LGA compared with the AGA group. Studies, having shown high expression of chemerin mRNA in mature adipocytes and adipose tissue of obese animals, suggest that chemerin levels reflect the adipocyte cell size and the total body fat mass [11]. Furthermore, in previous reports, circulating levels of chemerin were significantly associated with metabolic syndrome parameters (circulating triglycerides, blood pressure, body fat content, and insulin resistance) [11]. Therefore, the increased fat mass of the LGA fetuses might explain the elevated fetal chemerin levels in this group. Moreover, increased chemerin levels possibly imply the predisposition to insulin resistance and thus, may serve as an early prognostic marker for development of metabolic syndrome in the future.

Animal studies have reported obestatin to decrease food inges-tion and body weight, as opposed to ghrelin, which increases food intake and weight [14,24,25]. In addition, human studies have demonstrated significantly lower circulating obestatin concentrations in obese and obese diabetic pregnant women compared with controls [26]. Furthermore, decreased obestatin levels were found in patients with T2DM and impaired glucose regulation, suggesting that body fat mass and insulin sensitivity possibly affect obestatin concentrations [13]. Interestingly, there are conflicting data on whether obestatin has a stimulating effect [27], an inhibiting effect [28], or no effect at all [29] on insulin secretion. It is postulated that obestatin exerts a dual effect on glucose-induced insulin secretion: at a low concentration, it potentiates insulin response to glucose, while at a high concentra-tion, it inhibits insulin release evoked by this stimulus [27]. This could partly explain the lack of significant correlation between insulin and obestatin in the LGA group of the present study.

Our results did not show significant differences in obestatin levels between LGA and AGA fetuses. Since obestatin levels are related to insulin resistance and glucose intolerance, the lack of difference in obestatin levels between the two groups prob-ably reflects lack of insulin resistance in LGA fetuses. The exact onset of insulin resistance in the metabolic syndrome prone LGA

Figure 2. Obestatin levels in umbilical cord serum of appropriate for gestational age (AGA) and large for gestational age (LGA) fetuses. Box plots represent median and interquartile range.

Figure 3. Insulin levels in umbilical cord serum of appropriate for gestational age (AGA) and large for gestational age (LGA) fetuses. Box plots represent median and interquartile range.

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individuals is not fully elucidated, although data suggest that it occurs later in life and not at birth [30]. In contrast, chemerin reflecting the adipocyte cell size and the total body fat mass is increased in LGA fetuses, thus, explaining the lack of significant correlation between obestatin and chemerin at birth, found in the present study.

The contribution of the placenta in the production and transfer of the above adipokines could not be documented in this study.

In conclusion, the increased adipose tissue in cases of LGA, might account for the elevated chemerin levels in this group, which could possibly serve as a prognostic marker for the later development of metabolic syndrome. On the other hand, the lack of difference between AGA and LGA fetuses in circulating levels of obestatin–possibly a sensitive marker of insulin resistance–might be due to development of metabolic disorders later in life and not present at birth. However, since we are just beginning to understand the role of these adipokines, further studies are needed to elucidate their interaction in the perinatal period.

Declaration of Interest: The authors report no conflicts of interest.

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