Longitudinal changes in leptin and adiponectin concentrations through uncomplicated pregnancy

Highlights:

• The highest concentrations of leptin are found in the second half of pregnancy, whereas adiponectin levels show a tendency to decline over pregnancy

• Adiponectin might be part of the protective systems that counterbalance a transient proatherogenic state observed in pregnancy mainly by improving the HDL levels

• Leptin might enhance bone formation in late pregnancy

Introduction

Normal pregnancy is characterized by profound metabolic and biochemical changes, to meet the needs of the growing fetus and to prepare mother for labor and lactation. One of the most prominent features of pregnancy is insulin resistance, especially in late pregnancy. It ensures the energy needs of the fetus and placenta are achieved, by means of transferring the glucose from maternal tissues to the fetus. This is quickly reversed after the delivery [1]. Adipose tissue morphology and function undergo extensive changes during pregnancy, even in nonobese, normoglycemic healthy women [2]. The adipose tissue is not only a storage depot of excess energy, it also produces adipokines that show numerous endocrine and paracrine effects. They play an important role in the metabolic homeostasis during normal gestation and complications of pregnancy [3]. The most studied are leptin and adiponectin.

Leptin is an adipokine that is implicated in energy metabolism, food intake and appetite regulation, inflammation, immune response, reproduction and angiogenesis [4]. It is mainly produced by adipose tissue. It is also produced by the placenta during pregnancy. In pregnancy, leptin has pleiotropic effects that are likely to influence implantation, placental angiogenesis and immunomodulation. Leptin is also involved in the fetal-maternal dialog, modulating the maternal-to-fetal transport of nutrients, particularly amino acids and lipids, thus contributing to adaptations to fetal nutritional requirements [5].

Adiponectin is an adipokine that is inversely related to insulin resistance, atherosclerosis, type 2 diabetes mellitus, hypertension, dyslipidemia, metabolic syndrome, hyperuricemia, non-alcoholic fatty liver disease, etc. [6]. Adiponectin receptors are expressed in human placenta, but there is no evidence of adiponectin expression by the placenta [7].

Low 25-hydroxyvitamin D [25(OH)D] and adiponectin levels are associated with obesity and cardiovascular disease, and there is evidence of positive correlation of adiponectin and 25(OH)D in the general population [8]. Osteocalcin (OC) is a bone-derived hormone which affects glucose metabolism by regulating insulin secretion and sensitivity. These extraskeletal actions of osteocalcin might be mediated by adiponectin [9]. It is also released into the circulation during bone formation, along with procollagen type 1 N-terminal propeptide (P1PN).

The aim of this study was to explore the longitudinal changes of leptin and adiponectin in pregnancy as well as their associations with lipid profile, insulin and bone formation parameters in late pregnancy.

Materials and methods

Women were enrolled at their first prenatal visit to the laboratory of Poliklinika Medilab, Čačak, Serbia. Trained staff recorded a full medical history from each participant, including preexisting disorders, smoking status and nutritional habits. They were all non-smokers and were given advice on balanced diet, which included six to 11 servings of bread and grains, two to four servings of fruit and three servings of protein sources daily. All women used vitamin supplementation which contained approximately 400 μg of folic acid, 400 IU of vitamin D, 200–300 mg of calcium, 70 mg of vitamin C, 3 mg of thiamine, 2 mg of riboflavin, 20 mg of niacin, 6 μg of vitamin B12, 10 mg of vitamin E, 15 mg of zinc and 17 mg of iron. The exclusion criteria were non-singleton pregnancy or the development of any complications during pregnancy, including gestational diabetes mellitus. They underwent an oral glucose tolerance test with 75 g of glucose between weeks 24 and 28 and showed normal glucose tolerance according to the National Guidelines for Diabetes Mellitus [10].

In total, 38 women completed the study; 21 of them were primiparas and 17 were multiparas. The median of age was 30 years (range 20–37). All of them had an uneventful pregnancy and full-term delivery. All infants were healthy and had birth weights between the 10^th and 90^th percentiles.

The study was planned and conducted according to the ethical standards of the Declaration of Helsinki and Local Institutional Guidelines. The local Institutional Review Committee approved the research proposal. Informed consent was obtained from all individuals involved in the study.

Body mass index (BMI) was calculated as weight (kg)/squared height (m²).

The samples from each participant were taken at five time points: in the 1^st trimester (range 11–13 weeks, T1), in 2^nd trimester (range 22–24 weeks, T2), at the beginning of the 3^rd trimester (range 27–29 weeks, T3), later in the 3^rd trimester (range 35–37 weeks, T4) and more than 4 weeks postpartum (T5). The sampling was performed after an overnight fast (>10 h), between 7 am and 9 am, in a serum test tube. The samples were centrifuged and for the delayed analyses, the sera were aliquoted and stored at −80 °C within 1 h of sampling. The samples were thawed only once, immediately before analysis. The measurements of glucose, total cholesterol (t-C), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-C), uric acid and C-reactive protein (CRP) were performed immediately.

Glucose, t-C, TG, HDL-C, uric acid and CRP were measured using commercial kits on Roche Cobas c311 clinical chemistry analyzer (Roche Diagnostics, Mannheim, Germany). Insulin, 25(OH)D, N-MID OC, and P1NP were measured by electrochemiluminescence immunoassay (ECLIA) on the Cobas e411 analyzer (Roche Diagnostics, Mannheim, Germany). The intra- and inter-assay coefficients of variance were as follows: for insulin 2.0% and 3.9%, respectively, for 25(OH)D 2.7% and 5.2%, for N-MID OC 2.0% and 4.1%, and for P1NP 2.1% and 4.9%. The Roche N-MID OC assay detects stable N-MID-fragment as well as the intact osteocalcin, and it remains unchanged after 3 h at room temperature and after 24 h at 4 °C [11].

Low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald equation:

LDL-C (mmol/L)=TC (mmol/L)−HDL-C (mmol/L)−TG (mmol/L)/2.22

Total plasma adiponectin concentration was measured in duplicate and assayed by an enzyme-linked immunosorbent assay (ELISA) method (Human Adiponectin/Acrp30 Immunoassay, Quantikine, R&D systems, Minneapolis, MN, USA). The concentration of leptin in plasma was determined using a Leptin (sandwich) ELISA Kit (DRG Instruments, Marburg, Germany). All of the samples were measured in the same batch, immediately after the thawing. For adiponectin, the intra- and inter-assay coefficients of variance were 3.7% and 6.8%, respectively. For leptin, the intra- and inter-assay coefficients of variance were 3.1% and 6.9%, respectively.

Results

General characteristics and biochemical parameters of the study group are shown in Table 1. t-C levels showed a progressive increase until the 3^rd trimester, with a post-partum decline, where the post-partum t-C level was still significantly higher than the 1^st trimester value. LDL-cholesterol showed the same increasing pattern during pregnancy. After delivery, its value remained higher than the 1^st and 2^nd trimester values (Table 1). TG levels also demonstrated a progressive increase, with the T4 value even higher than the T3 value. There was a more pronounced decrease after the delivery and the postpartum values were somewhat similar to the 1^st trimester value. HDL-cholesterol increased in the 2^nd and early 3^rd trimester in comparison to the 1^st trimester and dropped after delivery to a value lower than all of the pregnancy values (Table 1). All of the participants retained normal glucose level throughout pregnancy. Insulin levels showed a significant increase in the late 3^rd trimester, as expected, owing to increased insulin resistance in the late pregnancy. It then dropped to a value significantly lower than the values in T1 and T4 (Table 1). Uric acid increased progressively during pregnancy and continued to increase after delivery (Table 1). The levels of CRP showed the highest value in T2, and the lowest value after the delivery. Leptin concentrations showed an increase which reached statistical significance in T3 and T4 (3^rd trimester), and a significant decrease post-partum in comparison to the values in T3 and T4. Although the adiponectin concentrations showed a noticeable tendency to decline over pregnancy, there was no statistical significance until after the delivery. The post-partum value was significantly lower than the values in T1 and T2 (Table 1).

25(OH)D levels showed a significant increase in T2 and no change in T3, and a significant decrease in T4 and further decrease after delivery. Osteocalcin showed a decrease in the 2^nd trimester, but it increased markedly in T4 and even more strikingly postpartum (Table 1). P1NP increased from T3 and remained high in T4 and T5 (Table 1).

Spearman’s correlation analyses were performed to test for associations between leptin/adiponectin and other investigated parameters in the late 3^rd trimester. (Tables 2 and 3). Leptin was significantly positively correlated with BMI, uric acid, insulin, OC and P1NP (all p<0.05), and CRP (p<0.001) (Table 2). Adiponectin was positively correlated with HDL (p<0.05), and negatively with BMI, glucose, OC (all p<0.05), and TG and insulin (p<0.001) (Table 3).

Multiple regression analysis was performed to find which of investigated parameters was independently associated with adiponectin in the 3^rd trimester. The result of this analysis showed that HDL is independently associated with adiponectin level during the 3^rd trimester (p<0.05) (Table 4).

Discussion

In this study, we observed a significant increase in leptin concentrations in the second half of pregnancy. Previous studies have demonstrated that maternal leptin levels are elevated during pregnancy and suggest that leptin concentrations peak around 28 weeks of gestation and decrease to pregravid concentrations after delivery [4].

The gestational increase in leptin is thought to originate from the placenta rather than from the adipose tissue. This is because the rise of leptin precedes the physiological increase in maternal BMI during pregnancy, it is not correlated with adiposity, and circulating leptin levels decline drastically within 24 h after delivery [5]. A positive energy balance should be maintained during pregnancy. As a result, it is unlikely that the increased leptin concentrations reduce the food intake by its central action. In this setting, they suggest different roles of leptin or they might reflect a state of leptin resistance [14].

Leptin is already known as a proinflammatory adipokine because it can enhance the production of several proinflammatory cytokines (IL-6, IL-12, IL-18, TNFα, etc.) in peripheral blood monocytes, it can also induce the production of reactive oxygen intermediates in macrophages, neutrophils, and endothelial cells, enhance platelet aggregation and promote leukocyte-endothelial cell interactions [15]. We found positive correlations of leptin with CRP in the 3^rd trimester, which is concordant with the pro-inflammatory properties of leptin. Leptin also showed a positive correlation with insulin which is in line with previous studies [16].

Human chorionic gonadotropin is likely to increase placental leptin synthesis and secretion, whilst the increase of estradiol levels during gestation is likely to maintain leptin production. In addition, elevated levels of prolactin and placental lactogen may directly interfere with leptin receptor signaling possibly predisposing the mother to leptin resistance [5].

Observed changes in t-C, TG, HDL- and LDL-cholesterol are in line with previous studies [1, 17]. These changes promote accumulation of maternal fat stores in early pregnancy and fat mobilization in late pregnancy [1]. The increasing concentrations of estrogen and insulin resistance lead to hypertriglyceridemia in late pregnancy. It is a consequence of increased lipolysis and enhanced flux of fatty acids to the liver and subsequent synthesis and secretion of very low density lipoprotein (VLDL) particles into the circulation. Also, the activity of lipoprotein lipase is reduced, so VLDL particles remain in circulation longer and lead to higher production of LDL particles [18]. These changes lead to a more atherogenic profile in the late pregnancy. Additionally, the HDL concentration increases from the 2^nd trimester, but its increase is not as pronounced as LDL. HDL has anti-atherogenic effects, through its role in reverse cholesterol transport and its anti-inflammatory and antioxidant activities [19]. These metabolic changes in pregnancy are short-term and reversible and therefore it is unlikely that they induce permanent changes in the cardiovascular health of otherwise healthy women, with no complications during pregnancy.

In our study, the highest level of adiponectin was found in the first trimester. Although noteworthy, the decrease in adiponectin did not reach statistical significance until after the delivery (Table 1). A study by Fuglsang et al. [20] found increasing levels of adiponectin in the first part of pregnancy, followed by declining levels throughout pregnancy from maximum levels at mid-gestation. Another study showed similar results, with the adiponectin levels decreasing only after the 3^rd trimester and reaching the lowest levels after the delivery and also after 1 month [21]. However, Mastorakos et al. [16] found no significant change of adiponectin during pregnancy. The initial increase in adiponectin concentrations cannot be readily explained by the increase in BMI. Maternal fat gain is highest in the 2^nd trimester when adiponectin levels start to decline [20]. An in vitro experiment on human adipocytes suggested that prolactin may regulate the production of adiponectin by adipocytes [21], so the decrease in adiponectin concentrations does not appear to be attributable to central fat accumulation and weight gain, but rather to the inhibitory effect of prolactin. This may explain the decline of adiponectin levels in all of the mentioned studies, including ours.

The anti-inflammatory effects of adiponectin are mediated through its receptors, which activate adenosine monophosphate (AMP) protein kinase in immune cells and tissues. The high-molecular form of adiponectin has anti-inflammatory properties that inhibit inflammation by blocking nuclear factor NF-κB activation and reducing such cytokines as TNFα, IL-6 and IL-18 [15]. However, in our study adiponectin and CRP did not show a significant correlation in the 3^rd trimester.

Fuglsand et al. [20] did not observe any significant associations between adiponectin and insulin levels, and in another study, adiponectin concentrations did not correlate with markers of insulin sensitivity during pregnancy [16]. We found a significant negative correlation of adiponectin and insulin, as did Catalano et al. [22]. However, Ritterath et al. [23] found no correlation between adiponectin and carbohydrate metabolism, but they found a negative correlation of adiponectin with TG levels and positive correlation with HDL-cholesterol during pregnancy. In our study, adiponectin was positively correlated with HDL-cholesterol and negatively correlated with TG in the late pregnancy (Table 3). Adiponectin influences plasma lipoprotein levels by altering the levels and activity of key enzymes (lipoprotein lipase and hepatic lipase) responsible for the catabolism of TG-rich lipoproteins and HDL [24].

In our study, leptin was positively correlated with both OC and P1NP (Table 2), which was in line with the observation that leptin increases osteoblast number and activity thus enhancing bone formation [25]. Adiponectin was negatively correlated with OC, perhaps reflecting a compensatory mechanism of OC action on the state of decreasing adiponectin, which could be regarded in the light of experimental data obtained on adipocytes, that found that OC increased secretion of adiponectin [9]. Srichomkwun et al. [26] found no association between OC and adiponectin levels in pregnant women. As P1NP showed no significant correlation with adiponectin, it can be speculated that the metabolic effects are specific for OC, and not the other products of bone formation, such as P1NP. Taken together with previous data that suggest a potential compensatory role of OC on insulin resistance in pregnancy [27], these findings further confirm complex interrelationships between bone, glucose metabolism and adipose tissue.

Neither leptin nor adiponectin levels were correlated to 25(OH)D (Tables 2 and 3). No significant relationship existed between maternal or fetal leptin and 25(OH)D in the study of Walsh et al. [28]. In the general population, there was no correlation between adiponectin and 25(OH)D [29]. These findings suggest that potential extra-skeletal effects of vitamin D are not straight forward or readily explained.

The result of multiple regression analysis showed that only HDL concentration is independently associated with adiponectin level during 3^rd trimester and it independently explained 28.1% of variance in adiponectin concentration. Similar independent positive correlations were observed in non-pregnant individuals [30]. Adiponectin has a number of possible roles including stimulating the activity of peroxisome proliferation activated receptor α ligand (PPARα) in both skeletal muscle and liver, acting as an important component in the metabolism of apo AI by reducing its catabolism, and also reducing hepatic lipase activity [31]. It appears that adiponectin, especially in its high-molecular form, could act to prevent cardiovascular disease both through the effects on the vascular wall and by promoting a more favorable lipoprotein subclass profile as it is shown to be negatively correlated with serum triglycerides and positively with HDL particles. Similar correlations of adiponectin and lipid status parameters were observed in other studies [32, 33]. Data from in vitro studies suggest that adiponectin reduces expression of adhesion molecules in endothelial cells and decreases cytokine production from macrophages by inhibiting the NF-κB signaling, suppresses macrophage to foam cell transformation, and also proliferation and migration of smooth muscle cells induced by platelet-derived growth factor [34, 35]. These effects of adiponectin on the vascular wall were confirmed by clinical observations that indicate a close relationship between hypoadiponectinemia and peripheral arterial dysfunction [36].

The results of our study suggest complex interactions of leptin and adiponectin with glucose and lipid metabolism during pregnancy. These adipokines might have a role in the development or regulation of physiological insulin resistance in pregnancy. Adiponectin might be part of the protective systems that counterbalance a transient proatherogenic state observed in pregnancy mainly by improving the HDL levels. Furthermore, the observed correlations of adiponectin and leptin with the bone formation parameters support the notion that adipose tissue, bone, glucose and energy metabolism are interrelated. The exact mechanisms and potential implications in pathological states, such as gestational diabetes, remain unexplained and require further investigation.