Our aim was to study the expression of adipokine-encoding genes (leptin, adiponectin, and angiopoietin-like protein 4 (ANGPTL4)) in human umbilical vein endothelial cells (HUVECs) and adipokine concentration in cord blood from women with gestational diabetes mellitus (GDM) depending on glycaemic targets. GDM patients were randomised to 2 groups per target glycaemic levels: GDM1 (tight glycaemic targets, fasting blood glucose < 5.1 mmol/L and <7.0 mmol/L postprandial,
The intrauterine hyperglycaemia in women with gestational diabetes mellitus (GDM) is supposed to be an important factor that predisposes offspring to obesity and type 2 diabetes mellitus (T2D) [
There is some suggestion that the exposure to diabetes in utero increases the risk of offspring obesity via alterations in the “adipoinsular axis,” the endocrine loop, linking the brain and endocrine pancreas with insulin- and leptin-sensitive tissues in the control of eating behaviour and energy balance [
Adipokines play an important role in the energy metabolism regulation [
Another promising adipokine is angiopoietin-like protein 4 (ANGPTL4), a multifunctional signal protein expressed in many tissues. ANGPTL4 is involved in the regulation of multiple physiological processes, including energy metabolism [
The change in the expression of the abovementioned genes in the fetal tissues may serve as a marker of subsequent metabolic diseases of the offspring. The association between the presence of hyperglycaemia in the mother and altered cord blood levels of leptin, adiponectin, and ANGPTL4 has been identified in previous studies [
However, it is not obvious that maternal hyperglycaemia causes such alterations. Perhaps, on the contrary, the altered gene expression functions in GDM pathogenesis (e.g., due to activation of hormone-encoding genes evoking insulin resistance or reduction of insulin secretion). It is also possible that both phenomena (maternal hyperglycaemia and changes in the expression of adipokines in the fetus and/or the mother) result from other pathological processes.
Randomised controlled trials (RCT) comparing changes of newborn gene expression level in groups of women with different target glucose levels during the treatment of GDM are supposed to help clarifying the cause-and-effect relations.
The human umbilical vein endothelial cells (HUVECs) represent a good cellular model for studying the effect of maternal hyperglycaemia on the fetal cardiovascular system and can serve as a marker of the predisposition of the fetus to metabolic diseases [
In this study, we investigated the alterations in ANGPT4, ADIPOQ, LEP, and leptin receptor gene (LEPR) expression levels in HUVECs and concentrations of these adipokines in the cord blood from newborns of women with GDM with different glycaemic targets compared to healthy women.
This study was carried out at the Almazov National Medical Research Centre (ANMRC) as part of the ongoing RCT “Genetic and epigenetic mechanisms of developing gestational diabetes mellitus and its effects on the fetus” (GEM GDM) which started in July 2015. This study was approved by the local ethical committee (Protocol 119); informed written consent was obtained from all subjects.
Forty-one women with GDM and 25 controls were randomly selected to assess the levels of expression of genes in HUVECs. The women with GDM were randomised to 2 groups according to target glycaemic levels: group 1 (target fasting blood glucose < 5.1 mmol/L and <7.0 mmol/L 1-hour postprandial) (GDM1,
GDM was diagnosed according to the Russian National Consensus [
None of the patients had previous history of diabetes mellitus or any known medical condition affecting glucose metabolism.
They were all followed until delivery at ANMRC. Anthropometric variables (height and blood pressure) were measured using standardised procedures. Prepregnancy body mass index (BMI) was calculated based on the prepregnancy weight recalled by participants. Women with GDM were consulted by the endocrinologist and provided the results of their self-measurements of blood glucose every 2-3 weeks. In case of exceeding the target blood glucose levels (in 2 or more measurements per week in group 1 and in more than 1/3 of measurements per week in group 2), insulin therapy was started. The participants were asked to keep electronic nutrition and glycaemic control diaries with the help of a specially developed mobile application and send data to the doctor. The mobile application is described elsewhere [
Cord blood samples were collected immediately after delivery. Blood glucose measurements were made on fresh plasma samples. The cord blood serum samples were stored at −80°C for further analysis of C-peptide, leptin, adiponectin, and ANGPTL4. Plasma glucose (PG) concentration was determined by the glucose oxidase method. Serum C-peptide level was measured by the chemiluminescent microparticle immunoassay (Architect C-peptide assay, Abbott Laboratories, IL, USA). Serum adiponectin (BioVendor Laboratory Medicine Inc., Modrice, Czech Republic) and leptin (Diagnostics Biochem Canada Inc., Canada) levels were measured using an enzyme-linked immunosorbent assay (ELISA) as recommended by the manufacturer. Serum level of ANGPTL4 was determined by DuoSet ELISA Development kits (DY3485) from R&D Systems (USA). The limit of detection for ANGPTL4 is 1.25 ng/mL. The detection range is 1.25 ng/mL–80 ng/mL. The following factors prepared at 800 ng/mL were assayed and exhibited no cross-reactivity or interference: recombinant human angiopoietin-1, angiopoietin-2, angiopoietin-4, and angiopoietin-like 3 and recombinant mouse angiopoietin-3 and angiopoietin-like 3.
The limit of detection for Leptin is 0.5 ng/mL. The detection range is 0.5–100 ng/mL.
The following substances were tested at 1000 ng/mL and exhibited no cross-reactivity: mouse leptin, TNF-α, IL-2, IL-3, IL-4, IL-6, IL-8, IL-9, IL-10, IL-12, IL-16, GM-CSF, CSF, and EGF.
The limit of detection for adiponectin is 26 ng/mL. The detection range is 26 ng/mL–100 ug/mL. No cross-reactivity has been observed for human leptin and leptin receptor.
Intra-assay coefficients of variation (CVs) for leptin assay were between 3.7% and 5.5%, and interassay CVs were 5.8–6.8%. For adiponectin assay, the intra- and interassay CVs were 3.9–5.9% and 6.3–7.0%, respectively.
The HUVECs were isolated using a standard collagenase digestion method [
The purity of primary HUVEC cultures was evaluated by flow cytometry analysis performed on Guava EasyCyte8. Briefly, detached cells were resuspended in 200
The viability of HUVEC was assessed by flow cytometry with the determination of the number of viable cells, as well as those in early and late apoptosis and necrosis evaluated by Annexin-V/PI (BioLegend, San Diego, CA, USA) double staining.
The expression of von Willebrand factor and CD146 (BioLegend, San Diego, CA, USA) in HUVECs was detected by immunocytochemical staining. Cell nucleuses were stained with 4
Total RNA was extracted from HUVEC using ExtractRNA reagent (BC032, Evrogen, Moscow, Russia) according to the manufacturer’s protocol. One microgram of total RNA was reverse transcribed using Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) kit (SK021, Evrogen, Moscow, Russia). After cDNA synthesis, quantitative real-time PCR was performed in 25
Relative expression was evaluated according to the 2−ΔΔCt method [
Statistical analysis was performed using SPSS 22.0 (SPSS Inc., USA). Mean and standard deviation are reported for continuous variables, and numbers and percentages are reported for categorical variables. Differences among the groups were analysed by Mann–Whitney test (for comparison between two groups), Kruskal-Wallis test (for comparison of more than two groups) or chi-square test. A
Baseline characteristics of the participants are described in Table
Characteristics of the participants at study entry.
GDM 1 |
GDM2 |
Control |
|||||
---|---|---|---|---|---|---|---|
Maternal age, years | 30.9 ± 5.4 | 32.3 ± 5.0 | 30.8 ± 4.2 | 0.566 | |||
Prepregnancy BMI, kg/m2 | 25.4 ± 7.2 | 26.1 ± 6.5 | 23.4 ± 4.2 | 0.287 | |||
BP syst, mmHg | 120 ± 13 | 118 ± 12 | 112 ± 14 | 0.114 | |||
BP diast, mmHg | 76 ± 8 | 73 ± 10 | 69 ± 8 | 0.016 | 0.003 | 0.155 | 0.195 |
Fasting PG, mmol/L | 5.1 ± 0.8 | 5.0 ± 0.6 | 4.5 ± 0.4 | 0.007 | 0.004 | 0.003 | 0.396 |
OGTT 1 h PG, mmol/L | 10.2 ± 1.4 | 9.9 ± 1.6 | 6.9 ± 1.9 | <0.001 | <0.001 | <0.001 | 0.454 |
OGTT 2 h PG, mmol/L | 8.0 ± 1.6 | 8.8 ± 1.6 | 5.9 ± 1.5 | <0.001 | <0.001 | <0.001 | 0.231 |
Fasting leptin, ng/mL | 22.2 ± 20.7 | 29.5 ± 26.2 | 26.6 ± 17.0 | 0.561 | |||
Fasting adiponectin, ng/mL | 7.2 ± 3.3 | 9.1 ± 3.3 | 8.9 ± 2.5 | 0.077 |
Note: BMI: body mass index; BP: blood pressure; PG: plasma glucose; OGTT: oral glucose tolerance test.
Mean levels of fasting, 1-hour postprandial and average blood glucose measured by the participants during the study are described in Table
Blood glucose data from electronic diaries and gestational weight gain.
GDM 1 |
GDM2 |
Control |
|||||
---|---|---|---|---|---|---|---|
Gestational weight gain, kg | 9.9 ± 4.9 | 9.5 ± 5.9 | 15.2 ± 7.8 | 0.006 | 0.023 | 0.023 | 0.970 |
BG average, mmol/L |
5.6 ± 0.3 | 5.9 ± 0.4 | 6.0 ± 0.5 | 0.004 | 0.110 | 0.893 | 0.005 |
Fasting BG, mmol/L |
4.7 ± 0.4 | 4.8 ± 0.3 | 4.7 ± 0.3 | 0.499 | 0.835 | 0.421 | 0.735 |
1 h postprandial BG, mmol/L |
5.9 ± 0.3 | 6.4 ± 0.5 | 6.5 ± 0.7 | 0.002 | 0.088 | 0.818 | 0.002 |
Number of BG measurements | 140 ± 78 | 147 ± 60 | 42 ± 21 | 0.001 | <0.001 | <0.001 | 0.946 |
% ( |
40% (8) | 29% (6) | N/A | 0.495 |
Note:
Pregnancy outcomes and biochemical markers in cord blood are shown in Table
Pregnancy outcomes, biochemical markers in the cord blood, and ANGPTL4 gene expression in HUVECs.
GDM 1 |
GDM2 |
Control |
||
---|---|---|---|---|
Gestational age at delivery, weeks | 39.2 ± 1.5 | 39.3 ± 1.0 | 39.7 ± 1.0 | 0.261 |
Caesarean section, % ( |
30% (6) | 19% (4) | 20% (5) | 0.723 |
Birth weight, g | 3572 ± 488 | 3584 ± 577 | 3513 ± 555 | 0.856 |
Height, cm | 52.1 ± 2.5 | 52.4 ± 2.3 | 52.1 ± 2.5 | 0.990 |
LGA, % ( |
20% (4) | 23% (5) | 12% (3) | 0.235 |
SGA, % ( |
5% (1) | 9.5% (2) | 4% (1) | 0.819 |
Apgar score 1 min | 7.5 ± 0.7 | 7.7 ± 1.1 | 7.7 ± 0.6 | 0.204 |
Apgar score 5 min | 8.6 ± 0.5 | 8.7 ± 0.9 | 8.8 ± 0.4 | 0.208 |
Glucose, mmol/L | 4.7 ± 1.2 | 5.3 ± 1.3 | 4.5 ± 1.2 | 0.203 |
C-peptide, ng/mL | 0.8 ± 0.5 | 1.0 ± 0.6 | 0.9 ± 0.4 | 0.379 |
Leptin, ng/mL | 8.8 ± 6.6 |
18.3 ± 16.1 | 10.6 ± 10.4 | 0.042 |
Adiponectin, ng/mL | 15.9 ± 11.5 | 16.3 ± 14.4 | 18.3 ± 14.3 | 0.843 |
LAR | 0.97 ± 1.31 | 1.70 ± 1.66 |
0.72 ± 0.46 | 0.038 |
ANGPTL4 in cord serum, ng/mL | 19.9 ± 15.0 | 14.1 ± 4.5 | 13.9 ± 5.2 | 0.248 |
ANGPTL4 relative expression in HUVECs | 23.1 ± 25.6 |
21.5 ± 25.8 |
98.3 ± 104.6 | 0.001 |
Notes: LAR: leptin/adiponectin ratio; LGA: large for gestational age; SGA: small for gestational age. LGA was defined by a birth weight exceeding the 90th percentile on standard charts. SGA was defined by a birth weight below the 10th percentile on standard charts.
There was no statistically significant difference among the groups in terms of pregnancy outcomes (percent of large for gestational age (LGA) and small for gestational age (SGA) newborns, delivery by caesarean section) and the level of C-peptide, adiponectin, and ANGPTL4 in cord blood serum and glucose in cord blood plasma. The level of leptin in cord blood serum was higher in the GDM2 group than in the GDM1 (
HUVECs were obtained from the umbilical vein, expanded in vitro, and characterised for expression of endothelial markers by flow cytometry and immunohistochemistry. All samples demonstrated characteristic endothelial morphology and immunophenotype CD45−/CD144+/CD31+/CD146+/CD105+ and stained positively for endothelial markers, von Willebrand factor, and CD146 (as demonstrated in our previous work [
ANGPTL4 expression was downregulated in the HUVECs derived from GDM patients compared to control group (23.11 ± 5.71, 21.47 ± 5.64, and 98.33 ± 0.92, respectively, for GDM1, GDM2, and control groups;
Gene expression analysis. (a) The level of relative ANGPTL4 mRNA expression in HUVECs from healthy (control) and GDM patients. (b) The level of relative ADIPOQ mRNA expression in multipotent mesenchymal stromal cells (MSC) (as negative control), adipocytes (as positive control), and HUVECs. (c) The level of relative LEPR mRNA expression in HUVECs from healthy (control) and GDM patients. (d) Correlation between the relative LEPR mRNA expression in HUVECs and the level of leptin in the cord blood.
We did not detect the expression of LEP in HUVEC but found out that they expressed LEPR, and the expression of LEPR demonstrated a decline in the GDM1/GDM2 groups compared to the control group, though the differences were not statistically significant (Figure
Our RCT has shown that GDM treated according to the most widely accepted current guidelines was associated with the increased LAR in cord blood and that LAR did not differ from the control group if GDM was treated aiming at tighter glycaemic targets. The GDM1 group (with tighter glycaemic targets) had lower levels of leptin compared to GDM2. We also found a decrease in the level of expression of ANGPTL4 in HUVEC of newborns from women with GDM in comparison with the control group. However, there was no difference in the level of expression of ANGPTL4 between the two groups of GDM with different glycaemic targets.
The most appropriate target levels of glycaemia for the management of GDM are not universally defined [
These targets were used by the Maternal-Fetal Medicine Units Network (MFMU) trial showing benefit for the treatment of GDM [
However, there are no reliable data from controlled trials of lower versus higher target levels of glycaemia to identify ideal glycaemic targets for prevention of fetal risks [
The glycaemic targets used in our study for group 1 were tighter in accordance with current Russian guidelines [
Follow-up studies are needed to understand the impact of tight glycaemic targets during pregnancy on obesity development in the offspring of women diagnosed with GDM according to IADPSG criteria. It is especially important taking into consideration the evidence that low early-life leptin concentrations may promote faster weight gain in infancy [
There is controversy about the association of adiponectin levels in cord blood with GDM. Pirc et al. reported decreased levels of adiponectin in the cord blood of newborns from mothers with GDM [
The level of expression of LEP in HUVECs turned out to be below the detection threshold. The expression of ADIPOQ was detected in HUVEC samples, but the level of its expression was as low as in negative control samples, which confirms that umbilical vein endothelium is not a place of adiponectin production. We are not aware of any other study addressing the expression of these genes in HUVEC. However, our results are in line with some of the previous studies which have shown that ADIPOQ is not expressed in the placenta [
In contrast to LAR changes associated with tight glycaemic targets of treatment of GDM, the level of expression of ANGPTL4 was lower in both the GDM groups regardless of glycaemic targets compared to the control group. Possibly, it is due to the fact that the difference in target glycaemic levels is not significant enough to affect the expression level of ANGPTL4, at least on such a small sample. Another plausible explanation is that the reduced level of activity of ANGPTL4 is transmitted at the genetic level to newborns from their mothers. Maybe, the reduced level of activity of ANGPTL4 contributes to the development of GDM in the mothers, that is, it is the cause, not the consequence of hyperglycaemia.
This hypothesis is supported by the data of Xu et al. on the lower level of ANGPTL4 in patients with type 2 diabetes whose pathogenesis is close to GDM [
Moreover, opposite to our data, Ortega-Senovilla et al. showed that serum ANGPTL4 concentrations in cord serum were higher in those from GDM than those from control pregnancies [
In addition, other factors besides intrauterine hyperglycaemia may affect the activity of a number of genes, including ANGPTL4, in the fetus. Known factors that affect the weight of the newborn are the body mass index (BMI) of the mother and maternal gestational weight gain. Obviously, these parameters are influenced by the mother’s lifestyle (the quantitative and qualitative composition of the diet and the level of physical activity).
It is known that ANGPTL4 can be regulated by diet [
This upregulation has been confirmed likewise in vitro as the expression of ANGPTL4 is upregulated in response to exposure to fatty acids in cell studies [
Our study established a significantly lower pregnancy weight gain in GDM patients compared to controls which is obviously due to diet adherence by patients.
Further studies are needed to clarify the cause-and-effect relationship between GDM and the level of expression of ANGPTL4 gene in HUVEC.
The level of C-peptide in the cord blood is commonly used as a marker of fetal hyperinsulinemia [
The strength of our study is the design of the RCT of different glycaemic targets for women with GDM which allows at testing cause-and-effect relationships. The weakness of the study, besides its relatively small sample size, is the lack of information about maternal levels of the studied gene expression.
Our study established positive association of cord leptin levels and LAR with target levels of glycaemia during pregnancy in women with GDM. Further investigation into long-term consequences of cord leptin concentrations is required.
We also found a decrease in the expression of ANGPTL4 in HUVECs of neonates from mothers with GDM. However, we could not prove the causal relationship between intrauterine hyperglycaemia and the expression of the ANGPTL4 gene, given the absence of differences between the level of expression of ANGPTL4 in groups with different glycaemic targets. This relationship remains to be clarified.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national guidelines on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008, and have been approved by the institutional committee of ANMRC (Protocol 119).
The authors declare no conflict of interest.
The study was funded by the Russian Science Foundation (Project no. 17-75-30052).