Cord blood irisin at the extremes of fetal growth

    Cord Blood Irisin at the Extremes of Fetal Growth Stavroula Baka, Ariadne Malamitsi-Puchner, Theodora Boutsikou, Maria Boutsikou, Antonios Marmarinos, Dimitrios Hassiakos, Dimitrios Gourgiotis, Despina D. Briana PII: DOI: Reference:

S0026-0495(15)00215-2 doi: 10.1016/j.metabol.2015.07.020 YMETA 53259

To appear in:

Metabolism

Received date: Revised date: Accepted date:

14 January 2015 19 July 2015 23 July 2015

Please cite this article as: Baka Stavroula, Malamitsi-Puchner Ariadne, Boutsikou Theodora, Boutsikou Maria, Marmarinos Antonios, Hassiakos Dimitrios, Gourgiotis Dimitrios, Briana Despina D., Cord Blood Irisin at the Extremes of Fetal Growth, Metabolism (2015), doi: 10.1016/j.metabol.2015.07.020

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CORD BLOOD IRISIN AT THE EXTREMES OF FETAL GROWTH

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Stavroula Baka1, Ariadne Malamitsi-Puchner1, Theodora Boutsikou1, Maria Boutsikou1, Antonios Marmarinos2, Dimitrios Hassiakos1†, Dimitrios

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Gourgiotis2, Despina D. Briana1

Department of Neonatology, Athens University Medical School, Athens, Greece

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Laboratory of Clinical Biochemistry-Molecular Diagnostics, 2nd Department of

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Pediatrics, Athens University Medical School, Athens, Greece †

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D.H. passed away

The authors state that they have no conflict of interest or financial support relevant to

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this article to disclose

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Short title: irisin in LGA and IUGR

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Corresponding author:

Ariadne Malamitsi-Puchner, MD 19, Soultani Street, 10682 Athens, Greece Tel: +30 6944443815, Fax: + 30 2107233330 E-mail addresses: [email protected], [email protected] Abbreviations: IUGR: intrauterine growth restriction, LGA: large for gestational age, AGA: appropriate for gestational age, WAT: white adipose tissue, BAT: brown adipose tissue, UCP-1: uncoupling protein 1, PGC-1α: peroxisome proliferatoractivated receptor-γ coactivator-1α, BMI: body mass index

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Abstract Background/Aims: Irisin, a novel myokine with antiobesity properties, drives

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brown-fat-like conversion of white adipose tissue, thus increasing energy expenditure

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and improving glucose tolerance. We aimed to investigate circulating irisin concentrations in large-for-gestational-age-(LGA) and intrauterine-growth-restricted-

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(IUGR) fetuses, both associated with metabolic dysregulation and long-term

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susceptibility to obesity and metabolic syndrome development. Methods: Plasma irisin and insulin concentrations were determined by ELISA and IRMA, respectively, in 80 mixed arteriovenous cord blood samples from LGA (n=30), IUGR (n=30) and appropriate-for-gestational-age (AGA, n=20) singleton full-

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term pregnancies. Fetuses were classified as LGA, IUGR or AGA, based on customized birth-weight standards adjusted for significant determinants of fetal

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growth.

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Results: Fetal irisin concentrations were lower in IUGR cases than AGA controls

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(p=0.031). Cord blood irisin concentrations were similar in LGA and AGA groups and positively correlated with birth-weight, as well as customized centiles (r=0.245, p=0.029 and r=0.247, p=0.027, respectively). Insulin concentrations were higher in LGA, compared to AGA fetuses (p=0.036). In the LGA group, fetal irisin concentrations positively correlated with fetal insulin concentrations (r=0.374, p=0.042). Conclusions: Impaired skeletal muscle metabolism in IUGR fetuses may account for their irisin deficiency, which may be part of the fetal programming process, leading to increased susceptibility to later metabolic syndrome development. Furthermore, irisin down-regulation may predispose IUGR infants to hypothermia at birth, by inducing

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less “browning” of their adipose tissue and consequently less non-shivering thermogenesis. Irisin upregulation with increasing birth-weight may contribute to a

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slower fat gain during early infancy (“catch-down”), by promoting higher total energy

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expenditure. The positive correlation between irisin and insulin in the LGA group may reflect a counterbalance of the documented hyperinsulinemia, which is partly

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responsible for the excessive fat deposition in the LGA fetus.

Key Words: irisin, intrauterine growth restriction, fetal macrosomia, fetus, insulin

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resistance

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1. Introduction Fetal growth disturbances, i.e. intrauterine growth restriction (IUGR) and fetal

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macrosomia, although usually caused by different pathologic conditions, are both

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associated with alterations in fetal adipose tissue development, permanent changes in the regulation of hormonal functions and a high tendency of individuals to develop

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obesity and related metabolic disorders later in life [1, 2]. IUGR fetuses present with a disproportionate reduction in fat mass, as compared with lean mass [3, 4], as well as

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with reduced growth and impaired development of skeletal muscle [3, 5]. Furthermore, IUGR neonates display enhanced insulin sensitivity at birth, followed by accelerated postnatal growth and subsequent emergence of insulin resistance [6]. On the other hand, large for gestational age (LGA) infants present with increased

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adiposity and reduced insulin sensitivity at birth, followed by higher rates of obesity

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and cardiometabolic diseases later in life [7, 8]. Irisin is a recently identified exercise-induced myokine, which triggers the conversion

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of white adipose tissue (WAT) to brown-like adipose tissue (BAT), leading to

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increased energy expenditure and, subsequently to improved tissue metabolic profile, by promoting weight loss, improved glucose tolerance, and insulin sensitization [9, 10]. Recent research suggests that irisin acts through up-regulation of uncoupling protein 1 (UCP-1) -which is expressed by BAT adipocytes- and induces release of chemical energy as heat, thus, protecting against hypothermia and obesity [9-12]. In this respect, irisin has recently attracted a lot of interest as a potential new target for the treatment of obesity and its associated disorders, since data suggest that irisin induces “browning” of WAT and BAT thermogenesis [13-16]. However, data regarding irisin in the perinatal period are scarce and regulation of circulating irisin in

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cord blood of well-characterized IUGR and LGA pregnancies has not been, to the best of our knowledge, assessed so far.

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The present case-control study was based on the hypothesis that cord blood irisin

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concentrations may differ between IUGR and LGA cases, compared with appropriate for gestational age (AGA) controls, since the former present with alterations in the

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development of fetal adipose tissue and skeletal muscle, metabolic dysregulation and

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increased susceptibility to later development of obesity and related metabolic disorders [1, 2]. Therefore, this study aimed to evaluate and compare cord blood irisin and insulin concentrations in IUGR, LGA versus AGA infants and investigate the

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2. Material and Methods

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association of the above concentrations with a variety of perinatal variables.

The study was conducted according to the Declaration of Helsinki. The Ethics

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Committee of our University Hospital approved the study protocol. Participating mothers provided signed informed consent before enrolment. The study population

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was partly previously described [17]. Briefly, eighty parturients giving consecutively birth either to 30 asymmetric IUGR (birth-weight ≤ 7th customized centile), 30 LGA (birth-weight ≥ 90th customized centile) and 20 AGA full-term singleton infants were prospectively recruited for this case-control study. The Gestation Related Optimal Weight (GROW) computer-generated programme was used to calculate the customized centile for each pregnancy [18]. Significant determinants of birth weight (maternal height and booking weight, ethnicity, parity, gestational age and gender) were entered to adjust the normal birth weight centile limits [18].

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Possible cause(s) of IUGR were maternal smoking > 10 cigarettes/day (n=9), thrombophilia under thrombo-prophylaxis with heparin (n=7), caffeine consumption

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(n=6), pregnancy-induced hypertension (n=3) treated with orally administered

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antihypertensives, hypothyroidism under supplementation therapy (n=2), gestational diabetes mellitus treated with diet (n=1), preeclampsia treated with intravenous

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hydralazine (n=1), and arterial hypertension treated with orally administered antihypertensives (n=1).

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Umbilical artery blood flow patterns and resistance indices (RI: maximal systolic velocity-maximal diastolic velocity/maximal systolic velocity) were recorded. An increased umbilical artery RI outside the normal range signified a pathologic flow

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pattern [19]. In all IUGR pregnancies blood flow was impaired, reflected by an increased umbilical artery RI [19]. However, no absent end-diastolic flow in the

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umbilical arteries was documented.

Amniotic fluid and placental weight were reduced in all IUGR cases [20, 21].

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Maternal gestational diabetes was recorded in 5 LGA pregnancies, all treated with

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diet. In the AGA group, mothers were healthy non-smokers and placentas were normal in appearance and weight [21]. All women were supplemented with iron, folic acid and calcium. Eight out of 30 mothers with IUGR offspring and 13 out of 30 mothers with LGA offspring were overweight/ obese, while in the AGA group, 3 out of 20 mothers were overweight and 17 out of 30 mothers had normal weight [22]. Pregnancies with chromosomal aberrations, fetal malformations, congenital or acquired infections and genetic syndromes were excluded. One- and five-minute Apgar scores were ≥ 8 in all neonates.

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Clinical characteristics of participating mothers and infants of each group are shown in Table 1.

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Mixed arteriovenous cord blood samples were collected in pyrogen-free tubes from

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the doubly-clamped umbilical cords at birth, reflecting the fetal state. Blood samples were transferred within 20 minutes of sampling in the laboratory into special

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containers on ice and were immediately centrifuged. The supernatant plasma was

less than 6 months until assay.

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separated into two equal aliquots and immediately stored in a deep freezer (-80 ºC) for

Following the above strict sample collection and maintenance criteria and according to irisin ELISA manufacturers’ guidelines, treatment of samples with protease

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inhibitor (Aprotinin) can be omitted from the procedure without any problems. The determination of plasma irisin concentrations was performed by ELISA (Phoenix

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Pharmaceuticals Inc, Karlsruhe, Germany). The minimum detectable concentration,

respectively.

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intra- and interassay coefficients of variation were 11 ng/ml, 4-6% and 8-10%,

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This specific ELISA kit has been validated by previous recent studies [23-25]. The antibodies used have been extensively validated using western blot and they showed to correctly detect spiked irisin. The assay itself includes a positive control working in the range of 40-110ng/ml. Our measurements gave mean values of positive control: 72.5ng/ml, 77.8ng/ml, 77.7ng/ml and 77.6ng/ml, which fall exactly in the middle of the positive control’s range. These specific antibodies are therefore highly accurate in detecting true levels of irisin. Plasma insulin concentrations were measured by IRMA (Immunotech a.s, Prague, Czech Republic). The minimum detectable concentration, intra- and interassay coefficients of variation were 0.5 μIU/ml, ≤ 4.3% and ≤ 3.4%, respectively.

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3. Statistical analysis Data regarding irisin and insulin were not normally distributed (Kolmogorov-Smirnov

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test); thus nonparametric procedures were applied to examine any differences among

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groups. One way Anova test was applied to examine any differences in normally distributed continuous variables across groups. Otherwise, Kruskall Wallis test was

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performed. Pearson’s chi square test was used to estimate differences between categorical variables. Pearson’s correlation coefficient was used, where appropriate,

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to examine any positive or negative correlations. In case of non-normally distributed variables, Spearman correlation coefficient was used to explore possible significant correlations. SPSS 20.0 (Chicago, IL) was used for all calculations. A p< 0.05 was

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considered statistically significant.

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4. Results

Determined median (ranges) values of fetal irisin and insulin concentrations in the

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three studied groups, as well as p for trend for irisin and insulin concentrations are presented in Table 1. Fetal irisin concentrations were lower in IUGR cases, compared to AGA controls (p=0.031) (Fig. 1). Cord blood irisin concentrations were similar in LGA cases, compared to AGA controls (Fig. 1) and positively correlated with birth-weight, as well as customized centiles of the studied infants (r=0.245, p=0.029 and r=0.247, p=0.027, respectively) in a combined group. Fetal insulin concentrations were higher in LGA cases, compared to AGA controls (p=0.036) (Fig. 1) and positively correlated with birth-weight, as well as customized

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centiles of the studied infants (r=0.257, p=0.021 and r=0.249, p=0.026, respectively) in a combined group.

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In the LGA group, fetal irisin concentrations positively correlated with fetal insulin

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ones (r=0.374, p=0.042) (Fig. 2).

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When removing the outliers, fetal irisin concentrations were lower in IUGR cases, compared to AGA controls and LGA cases (p=0.002 and p=0.003, respectively). Cord

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blood irisin concentrations were similar in LGA cases and AGA controls. Fetal insulin concentrations were lower in IUGR cases, compared to LGA cases (p=0.007), while no differences were observed between IUGR cases and AGA controls, as well as between LGA cases and AGA controls.

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Finally, no association was found between fetal irisin concentrations and maternal

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age, parity, gestational age, delivery mode, gender, maternal diabetes or maternal smoking, but larger studies are needed to confirm or expand these data given the

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5. Discussion

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relatively low number of subjects in this initial study.

The results of this study indicate that fetal irisin concentrations are lower in IUGR than AGA infants. Impaired IUGR skeletal muscle growth [3, 5] may account for the documented irisin deficiency in IUGR fetuses. This irisin downregulation could probably contribute to the well-known susceptibility of IUGR infants to hypothermia at birth [26], by inducing less “browning” of their already diminished adipose tissue [3] and consequently less non-shivering thermogenesis at birth. Irisin, which appears to be maternally inherited [27], has been shown to be released in response to both exercise and shiver response during cold exposure [9]. After birth,

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the neonate rapidly cools down in response to the relatively cold extrauterine environment [28]. In order to survive, the newborn must accelerate heat production

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via non-shivering thermogenesis in BAT, mediated by the expression of BAT tissue-

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specific UCP1 [29]. Thus, BAT is essential for ensuring effective postnatal adaptation and its growth during gestation is largely dependent on glucose supply from the

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mother to the fetus [29]. Therefore, it may be assumed that BAT depots are probably

mother and irisin deficiency.

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diminished in the IUGR fetus, secondary to both reduced glucose supply by the

Since the discovery of BAT in adult humans, there has been a dramatic resurgence in research interest regarding its role in the defense against obesity and obesity-

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associated disorders [29-31]. Furthermore, a reduction in early BAT has been proposed to perpetuate through the life cycle, leading to suppression of energy

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expenditure and ultimately to obesity and impaired glucose homeostasis [32]. In this

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context, irisin downregulation in IUGR fetuses may suggest a reduction of early BAT depots, predisposing to the later emergence of insulin resistance. Thus, irisin

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deficiency in the IUGR fetus may be one of the missing links in the potential molecular pathways, responsible for the increased postnatal susceptibility of IUGR individuals to insulin resistance, obesity and obesity-related metabolic disorders. In accordance, a recent study documented lower umbilical artery irisin levels in pregnancies complicated by as stated “idiopathic (with no apparent cause) IUGR” [33]. Our findings further suggest similar fetal irisin concentrations in LGA and AGA infants, probably indicating that irisin may not be directly implicated in the metabolic disturbances associated with fetal macrosomia [1, 2, 7, 8]. Accordingly, a recent study suggests no differences in cord blood irisin levels between patients with GDM and

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healthy pregnant women [34]. However, a positive correlation between cord blood irisin concentrations and birth-weight, as well as customized centiles of the studied

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infants was documented. This association may contribute to a slower fat gain in

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heavier neonates during early infancy, by promoting higher total energy expenditure [9, 10]. Previous data suggest that newborns with higher birth-weight have a slower

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weight and fat gain than newborns with lower birth-weight during early infancy, probably due to higher total energy expenditure in the former [35, 36]. LGA subjects

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without “catch-down” during childhood present with a less favorable cardiometabolic phenotype, represented by higher body mass index (BMI), central adiposity and higher diastolic blood pressure in early adulthood [8]. Therefore, it could possibly be

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speculated that irisin upregulation with birth-weight and customized centile may exert a beneficial metabolic effect during early infancy. By contrast, a recent study

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demonstrated lack of a correlation between cord blood irisin levels and birth-weight

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[37].

Finally, our results suggest a positive correlation between fetal irisin and insulin

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concentrations only in the LGA group, probably reflecting a counterbalance to the documented hyperinsulinemia, which is partly responsible for the excessive fat deposition in the LGA fetus [38]. In accordance, a recent study investigating circulating irisin levels over a broad spectrum of body weight, showed a positive correlation between irisin and insulin [39]. Furthermore, relevant recent investigations revealed that irisin is independently and positively associated with fasting insulin during pregnancy [40] and with fasting blood glucose in children, suggesting that this hormone may play a crucial role in glucose metabolism [41]. A major strength of the present report is that cord blood samples were collected from three different well-characterized groups of infants with normal, restricted and

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exaggerated fetal growth, allowing for a detailed study of the possible role of irisin in early life, as well as the possible influences of the fetal metabolic state on irisin

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concentrations. Limitations of the study include the small sample size, the inability to

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adjust irisin and insulin concentrations for birth-weight and methodological considerations regarding irisin assays, for which, however, quality assurance data

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power calculation was not performed.

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have been provided in the Material and Methods section. Furthermore, an a priori

In conclusion, the present data provide evidence that irisin may be an important metabolic factor from a very early age. The documented irisin deficiency in IUGR fetuses may be part of the mechanisms underlying their susceptibility on the one hand

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to hypothermia at birth and on the other to insulin resistance-associated metabolic disorders later in life. Irisin upregulation with birth-weight and customized centile

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could probably contribute to a favorable metabolic profile during early infancy.

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Lastly, the positive association between irisin and insulin only in LGA fetuses may possibly indicate a beneficial effect of irisin against excessive fat deposition in fetal

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macrosomia. These findings could have important translational consequences, since the regulation of fetal adipose tissue development is implicated in both postnatal adaptation and metabolic programming, predisposing to the later emergence of metabolic diseases in infants born at the extreme of fetal growth. Further studies are required to elucidate the physiological significance of irisin in early life.

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Funding

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Financial support: none

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Disclosure statement

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Conflicts of interest: none

Author contributions

study and writing the manuscript.

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Stavroula Baka: Participated in the development of the protocol, execution of the

Ariadne Malamitsi-Puchner: Had primary responsibility for protocol development,

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patient screening enrollment, outcome assessment and critically reviewing the manuscript.

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Theodora Boutsikou: Had responsibility for patient enrollment.

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Maria Boutsikou: Performed the statistical analysis of the data. Antonios Marmarinos: Contributed to the conduction of the laboratory

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measurements.

Dimitrios Hassiakos†: Had responsibility for patient enrollment and screening. Dimitrios Gourgiotis: Participated in the analytical framework of the study and performed laboratory determinations. Despina D. Briana: Had responsibility for data analysis and principally writing the manuscript.

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40. Ebert T, Stepan H, Schrey S, Kralisch S, Hindricks J, Hopf L, Platz M, Lossner U, Jessnitzer B, Drewlo S, Blüher M, Stumvoll M, Fasshauer M. Serum levels of irisin in gestational diabetes mellitus during pregnancy and after delivery. Cytokine 2014;65:153-8. 41. Al-Daghri NM, Alkharfy KM, Rahman S, Amer OE, Vinodson B, Sabico S, Piya MK, Harte AL, McTernan PG, Alokail MS, Chrousos GP. Irisin as a

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predictor of glucose metabolism in children: sexually dimorphic effects. Eur J

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Clin Invest 2014;44:119-24.

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AGA (n=20)

IUGR(n=30)

LGA (n=30)

P value

Birthweight (grams)

3375.0±266.85

2475.7±286.42

4179.3±239.3

<0.001

Gestational Age (weeks)

39.1±0.9

38.5±1.5

38.99±0.9

0.170

Customized centile

56.0 (78.0-35.0)

3.5(7.0-0.0)

Maternal age (years)

34.0±4.8

33.0±4.7

95.5(100.0-90.0)

<0.001

32.9±4.4

0.891

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Male 10(50.0) Female 10(50.0) Mode of delivery Vaginal 11(55.0) 9(45.0)

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Cesarean section

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Gender

Parity

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First 12(60.0)

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Variables

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No 20(100.0)

Yes 0(0.0)

0.018

10(33.3)

21(70.0)

20(66.7)

9(30.0) 0.030

7(23.3)

7(23.3)

23(76.7)

23(76.7) 0.177

22(73.3)

15(50.0)

8(26.7)

15(50.0) 0.149

29 (96.7)

25 (83.3)

1 (3.3)

5 (16.7)

Smoking

0.796 No 20(100.0) Yes 0(0.0)

25(83.3)

26(86.7)

5(16.7)

4(13.3)

IRISIN (ng/ml)

247.3(393.8-206.9)

222.2(713.5-161.6)

240.4(1098.9187.7)

0.026

INSULIN (mIU/ml)

4.92(11.71-0.50)

3.89(16.16-0.50)

7.44(78.86-0.93)

0.008

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Table 1. Demographic data of participating mother/ infant pairs. Fetal irisin and insulin concentrations in the appropriate for gestational age (AGA), intrauterine

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growth restricted (IUGR) and large for gestational age (LGA) groups. Continuous

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variables are presented as MeanSD/ Median (Range). Categorical variables are

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presented as N (%).

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Figure legends Figure 1. Cord blood (UC) irisin and insulin concentrations in the appropriate for

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gestational age (AGA), intrauterine growth restricted (IUGR) and large for gestational

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age (LGA) groups. Circles represent ouliers and stars represent extreme values Figure 2. Positive correlation between cord blood (UC) irisin and insulin

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concentrations in the large for gestational age (LGA) group

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Figure 1

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Figure 2

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