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Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Mitochondrial translocation of human telomerase reverse transcriptase in cord blood mononuclear cells of newborns with gestational diabetes mellitus mothers§ Ping Li a,b,c, Yu Tong b,c, Huiming Yang a, Shu Zhou e, Fei Xiong a, Tingzhu Huo a, Meng Mao b,c,d,* a Department of Pediatrics, West China Second University Hospital, Sichuan University, No. 17, People‘s South Road, Chengdu 610041, Sichuan Province, PR China b Laboratory of Early Developmental and Injuries, West China Institute of Woman and Children’s Health, West China Second University Hospital, Sichuan University, PR China c Key Laboratory of Obstetric & Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, PR China d Chengdu Women’s and Children’s Central Hospital, No. 1617, Riyue Avenue, Chengdu 610091, Sichuan Province, PR China e Department of Obstetrics, West China Second University Hospital, Sichuan University, No. 17, People‘s South Road, Chengdu 610041, Sichuan Province, PR China
article info
abstract
Article history:
Aims: To better understand the role of oxidative stress in fetal programming, we assessed
Received 23 June 2013
the hypothesis that the mitochondrial translocation of human telomerase reverse tran-
Received in revised form
scriptase (hTERT) could protect neonatal mitochondrial DNA (mtDNA) from oxidative
8 November 2013
damage during pregnancies complicated by gestational diabetes mellitus (GDM).
Accepted 21 December 2013
Methods: 26 GDM mothers and 47 controls and their newborns were enrolled. The plasma
Available online xxx
levels of 8-isoprostaglandin F2a in maternal and cord blood were measured to evaluate
Keywords:
hTERT in cord blood mononuclear cells (CBMCs). Finally, the relative mtDNA content was
oxidative stress. Western blotting was then used to assess the mitochondrial localization of Gestational diabetes
analyzed by real-time PCR.
Fetal programming
Results: GDM mothers and their newborns had significantly higher levels of oxidative stress
Mitochondrial hTERT
than controls. hTERT was localized in both the nuclei and mitochondria of CBMCs, and the
Oxidative stress
increased CBMC mitochondrial hTERT levels were significantly correlated with elevated oxidative stress in newborns. The neonatal mtDNA content in the GDM group was comparable to controls, and was positively correlated with mitochondrial hTERT levels in CBMCs, suggesting that mitochondrial hTERT in CBMCs may have a protective effect on neonatal mtDNA in GDM pregnancies.
§ This study was supported by the national ‘‘11th Five-Year Plan’’ to support science and technology project grants, Ministry of Sciences and Technology, China (2009BAI80B00). * Corresponding author at: Laboratory of Early Developmental and Injuries, West China Institute of Woman and Children’s Health, West China Second University Hospital, Sichuan University, PR China. Tel.: +86 13668171437; fax: +86 02885501698. E-mail address: [email protected] (M. Mao). 0168-8227/$ – see front matter # 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.diabres.2013.12.024
Please cite this article in press as: Li P, et al. Mitochondrial translocation of human telomerase reverse transcriptase in cord blood mononuclear cells of newborns with gestational diabetes mellitus mothers. Diabetes Res Clin Pract (2014), http://dx.doi.org/10.1016/j.diabres.2013.12.024
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Conclusions: This study is the first to suggest that the mitochondrial translocation of hTERT in CBMCs under heightened oxidative stress might protect neonatal mtDNA from oxidative damage in GDM pregnancies. This could be an in utero adaptive response of a fetus that is suffering from elevated oxidative stress, and could help our understanding of the roles of oxidative stress in fetal programming. # 2014 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
Gestational diabetes mellitus (GDM) is a common complication of pregnancy that affects 5–10% of pregnancies in Asian females [1,2]. GDM pregnancy significantly increases the risk of a number of short- and long-term adverse consequences for the fetus, the most significant of which is a predisposition to the development of metabolic syndrome and type 2 diabetes [3–5]. The fetus in a GDM pregnancy is exposed to sustained higher glucose concentrations until birth. To adapt to a GDM intrauterine environment, the pattern of fetal metabolism is altered, including changes in the fetal pancreas allowing the production of increased levels of insulin and epigenetic modifications [6,7]. However, when the environmental conditions improve rapidly after birth, the in utero protective effects of the fetal predictive adaptive responses may have adverse effects on the long-term health of the newborns [8,9]. Although the fetal adaptive programming of metabolic disease has been proposed for a long time, its underlying mechanisms remain elusive. Notably, elevated oxidative stress (OS) is considered to be a major contributor [10–15], since not only GDM females but also their newborns exhibit a heightened level of OS [16–19]. Furthermore, increased OS was also detected in children born during compromised pregnancies, and was potentially related to insulin resistance and obesity in later life [20,21]. OS was reported to alter epigenetic modification via oxidant molecules that act as signaling factors, as well as induce susceptible fetal pancreas dysfunction and subsequent insulin resistance in the offspring [10,15,22]. OS plays an important role in the fetal programming of metabolic disease, although the mechanisms behind these effects remain elusive. A better understanding of the actions of OS in the newborns of GDM mothers is important to identify new ways to reinforce the programming role of OS in metabolic disease. The mitochondrion is a critical organelle that regulates OS, and plays important roles in fetal growth and development [23– 25]. MtDNA is the intrinsic genome of mitochondria, and is responsible for encoding the majority of the respiratory chain enzymes. It is also considered to be an indicator of mitochondrial function [26]. Previous studies demonstrated that mtDNA was protected by mitochondrially localized telomerase reverse transcriptase (TERT) under increased OS. TERT is the catalytic subunit of the telomerase holoenzyme, which is a ribonucleoprotein responsible for the maintenance of telomeres to alleviate replicative senescence and genetic instability. In addition to the telomere-dependent functions of nuclear TERT, studies have suggested that TERT was also localized to the mitochondria [27–30]. Importantly, endogenous mitochondrial TERT is not an artifact of the overexpression of proteins associated with cellular transformation [30–32]. A mitochon-
drial targeting signal (MTS) in the N-terminal sequence of hTERT and two binding regions (ND1 and ND2) in mtDNA were found to play roles in the translocation of TERT from the nucleus to the mitochondria [27,30]. Of note, increased hTERT was exported to the mitochondria and exerted protective effects on mitochondrial function under increased OS [27–30]. In addition, mitochondrial hTERT allowed cancer cells to evade death and multidrug resistance by enhancing mitochondrial function and reducing levels of cellular ROS [33,34]. Collectively, experimental data in vitro and in vivo demonstrated that the increased mitochondrial translocation of hTERT induced by OS plays a putative role in the protection of mitochondrial function. Nevertheless, little information is available on newborns of GDM pregnancies. In this study, we investigated the mitochondrial translocation of hTERT under increased OS in newborns of GDM pregnancies, and assessed its correlation with neonatal mtDNA content. This will help us to better understand the roles of GDM-associated OS in fetal programming.
2.
Materials and methods
2.1.
Recruitment strategy and subjects
Twenty-six pregnant females with GDM (study group) and forty-seven healthy pregnant females without GDM (control group) as well as their newborns were finally included in the study. Pregnant females who underwent in vitro fertilization or with multiple pregnancies, inherited metabolic diseases, pregestational diabetes, and chronic hypertension were excluded. GDM was confirmed using the World Health Organization’s diagnostic criteria. Women who had any of the following were regarded as having GDM: a 1-h 50 g glucose challenge test (GCT) 7.8 mmol/L, fasting plasma glucose of 7.0 mmol/L, 1-h oral glucose tolerance test (75 g OGTT) of 10.0 mmol/L, or 2-h OGTT 8.5 mmol/L. All patients were recruited from the outpatient clinic during their third trimester in the Obstetrical Department of Southwest Second University Hospital affiliated with Sichuan University, China from 2009–2011. All the enrolled females also delivered in this hospital. The Regional and Institutional Committee of Science and Research Ethics of the West China Second University Hospital approved the study, and a written informed consent was obtained from all participants.
2.2.
Baseline data and blood sample collection
Maternal data concerning age, parity, gravidity, height, prepregnancy weight, pregnancy weight gain, family history of
Please cite this article in press as: Li P, et al. Mitochondrial translocation of human telomerase reverse transcriptase in cord blood mononuclear cells of newborns with gestational diabetes mellitus mothers. Diabetes Res Clin Pract (2014), http://dx.doi.org/10.1016/j.diabres.2013.12.024
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diabetes, a history of delivering macrosomia, or low birth weight were collected by a self-administered pregnancy nutrition and background questionnaire. Specialist clinicians collected the birth information of each newborn. Third trimester peripheral blood of pregnant women was drawn and partly centrifuged (3500 g for 10 min at 4 8C) within 30 min of collection. The plasma and blood cells were then frozen in separate aliquots at 80 8C until use. The rest of the whole blood was also stored at 80 8C. In addition, 35 mL venous cord blood was collected from the placental side of the cord after delivery and umbilical cord clamping. Thirty milliliters were used to isolate mononuclear cells (CBMCs) within 24 h, and the rest was processed as described for the maternal peripheral blood.
2.3.
Evaluation of oxidative stress
8-Isoprostaglandin F2a (8-iso-PGF2a), a promising oxidative stress marker, was detected in the plasma of maternal and cord blood using a commercially available Direct 8-iso-PGF2a ELISA kit (Enzo Life Sciences, Farmingdale, USA). A true reflection of both free and esterified isoprostane was measured following the manufacturer’s instructions.
2.4.
Isolation of cord blood mononuclear cells
CBMCs were isolated from the umbilical venous cord blood samples by Ficoll-Hypaque density gradient centrifugation, and stored in liquid nitrogen until analysis.
2.5.
Subcellular fractionation and Western blotting
Mitochondrial fractions were extracted from the CBMCs using the Mitochondria Isolation Kit for cells (Pierce/Thermo, Rockford, USA), and nuclear fractions were extracted with the Nuclear/Cytosol Fractionation Kit (BioVision, Mountain View, USA). The protein concentrations were determined using a Micro-BCA protein concentration determination kit (Pierce/ Thermo). Each type of protein sample was then adjusted to the same concentration and stored in separate aliquots at 80 8C until further analysis. Western blotting was performed to confirm that subcellular fractions were pure and uncontaminated, and to investigate the sub-cellular localization of hTERT. Antibodies used included monoclonal anti-VDAC1/Porin (Abcam, 1:1000; loading control for the mitochondrial fraction), polyclonal anti-Histone H2A.X (Abcam, 1:1000; nuclear fraction) and antihTERT (Abcam, 1:1000).
2.6.
Detection of human telomerase reverse transcriptase
Levels of hTERT in CBMCs were measured using a commercially available hTERT ELISA kit (GenWay Biotech, San Diego, USA). Total mitochondrial and nuclear proteins (1.0 mg/mL) were prepared prior to detection. The ELISA was then performed following the manufacturer’s instructions. The protein levels of hTERT were calculated by subtracting the absorbance value of the negative control from the sample, and normalizing to the positive control.
2.7.
3
Quantification of mtDNA content by real-time PCR
Total DNA was extracted from maternal peripheral and venous cord blood using the QIAamp DNA isolation kit for blood cells (Qiagen, Hilden, Germany). After quantification and adjustment of each genomic DNA concentration using Thermo Scientific Varioskan Flash (Thermo Scientific), samples were stored in separate aliquots at 80 8C until analysis. The copy numbers of mtDNA and nuclear DNA (nDNA) were determined using real-time quantitative polymerase chain reaction (RT-PCR) on a Bio-Rad C1000 thermal cycler equipped with the CFX96TM optical module. MT-RNR2 was selected as a mitochondrial target, and GAPDH as a nuclear target. The nuclear target was used to quantify nuclear DNA and normalize the amount of mtDNA per cell. The primer sequences MT-RNR2 forward 50 -CCAAACCCACTCCACCTTAC30 , and reverse 50 -TCATCTTTCCCTTGCGGTA-30 ; and GAPDH forward 50 -TTCAACAGCGACACCCACT-30 , and reverse 50 CCAGCCACATACCAGGAAAT-30 were reported previously by Pejznochova et al. [25]. Real-time PCR amplification was performed in 10 mL containing 2 SsoFastTM EvaGreen1 Supermix (Bio-Rad), 200 nM each primer, and 0.5 mL of each analyzed DNA sample at a concentration of 50 ng/mL. The PCR conditions were as follows: one cycle of 95 8C for 15 min, 40 cycles of 95 8C for 15 s and 60.6 8C for 30 s (MT-RNR2) or 60.8 8C for 15 s (GAPDH), and 72 8C for 30 s, followed by 72 8C for 5 min and a melting curve from 72 to 92 8C (with fluorescence readings every 1 8C/10 s). Two-fold serial dilutions of the genomic DNA (from 100 to 6.25 ng) were included in each run to generate the calibration curve. All samples were run in triplicate for each gene. The standard deviation of the mtDNA assay in triplicate repeats of different samples was 8%. Data were analyzed using the comparative Ct method, where Ct was the cycle number at which the instrument first detected fluorescence above background noise. The D cycle threshold (DCt) values of each sample was obtained by subtracting the values for the reference gene from the sample Ct, thus normalizing to nuclear DNA. For each experimental sample, the 2DCt was calculated, and data are presented as relative quantification. The slope of the calibration curve reflected the reaction efficiency, and each correlation coefficient was between 0.99 and 1.00. Melting curve analysis and electrophoresis were used to analyze reaction specificity.
3.
Statistical analysis
The SPSS1 statistical package, version 13.0 (SPSS, Inc., Chicago, Illinois) was used for statistical analysis. Quantitative data with normal distribution were expressed as the mean standard deviation (SD), and analyzed by the independent sample t-test. Non-normal quantitative data were expressed as the medians with interquartile ranges (IQR), and analyzed using Mann–Whitney U-test. The correlation between two variables was performed by Pearson correlation test or Spearman rank correlation test according to the data distribution. A two-tailed p < 0.05 was considered to be statistically significant.
Please cite this article in press as: Li P, et al. Mitochondrial translocation of human telomerase reverse transcriptase in cord blood mononuclear cells of newborns with gestational diabetes mellitus mothers. Diabetes Res Clin Pract (2014), http://dx.doi.org/10.1016/j.diabres.2013.12.024
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Table 1 – Clinical characteristic of enrolled mothers and their newborns.
Maternal data Age (years) Parity Gravidity Prepregnancy BMI (kg/m2) Weight gain (kg) Fasting plasma glucose (mmol/L) Family history of diabetes (%) Macrosomia history (%) Low birth weight history (%) Neonate data Gestational age at delivery (wks) Birth weight (g) Birth weight ratio (%) Microsomia (%) Male ratio (%) Cesarean section (%) Fasting plasma glucose (mmol/L)
GDM (n = 26)
Control (n = 47)
31.8 (29.8–37.4) 1.12 0.3 2.4 1.5 21.6 3.2 13.8 3.9 5.1 0.5 38.5 26.9 11.4
30.2 (28.8–32.2) 1.11 0.3 1.8 1.2 20.3 2.1 13.2 3.2 4.4 0.5 17.0 6.4 6.4
Ns Ns 0.00 0.04 Ns 0.00 0.01 0.04 Ns
38.7 1.2 3383.7 542.7 1.1 0.1 1.5 61.5 34.6 4.1 (1.7–4.5)
38.6 1.0 3347.8 370.4 1.0 0.2 2.1 42.6 28.9 3.3(2.9–61.9)
Ns Ns Ns Ns Ns Ns Ns
P
Note: Values are medians with interquartile ranges or mean SDs. Ns, no significance.
4.
Results
4.1.
Clinical characteristics
Twenty-six GDM mothers and forty-seven non-GDM controls and their newborns were enrolled in the study. The GDM and control groups were well matched for gestational age. The clinical characteristics of the mothers and newborns are shown in Table 1. The median age of GDM mothers was 31.8 years, which was slightly (but not significantly) older than non-GDM mothers (30.2 years). Although GDM females had higher gravidity than the controls, parity was similar between groups. In addition, GDM women had a higher pre-pregnancy BMI, but weight gain was similar in both groups. Fasting plasma glucose levels were significantly higher in GDM
mothers compared with control. A family history of diabetes and history of delivering macrosomia were also more frequent in the GDM group. However, no significant differences were found in birth weights of newborns in the two groups.
4.2. Higher levels of oxidative stress in GDM mothers and their newborns To evaluate oxidative stress in GDM mothers and their newborns, an ELISA kit was used to measure the plasma levels of 8-iso-PGF2a, a stable and specific marker of oxidative stress. Compared with controls, plasma levels of 8-iso-PGF2a were increased significantly in both the maternal blood of GDM females (9336.3 2919.9 vs. 7390.9 2211.1, p < 0.05) and in the cord blood of their newborns (5792.4 1088.6 vs. 4430.6 908.0, p < 0.05; Fig. 1). Consistent with previous studies [18,19,35], our data demonstrated that both GDM mothers and their newborns were suffering from increased oxidative stress compared with non-GDM controls.
4.3. Mitochondrial hTERT in CBMCs positively correlated with oxidative stress in cord blood
Fig. 1 – Plasma levels of 8-isoprostaglandin F2a (8-isoPGF2a), a stable and specific marker for oxidative stress, were increased significantly in both GDM women and their newborns compared with non-GDM controls; *p < 0.001 vs. control. Twenty-six separate GDM maternal and cord blood samples, and forty-seven separate control maternal and cord blood samples were measured in triplicate.
Previous studies reported endogenous mitochondrial hTERT, and oxidative stress could induce accumulation of mitochondrial hTERT by its translocation from the nucleus [29,31,36]. Therefore, we used western blotting to determine whether mitochondrial hTERT could be detected in CBMCs. Data revealed that hTERT was localized in both the mitochondria and nuclei of CBMCs (Supplementary Fig. 1). Next, ELISAs were performed to determine whether there were different levels of mitochondrial hTERT in CBMCs from newborns with and without GDM mothers. As shown in Fig. 2, the ratio of mitochondrial/nucleic hTERT in CBMCs was elevated significantly in the GDM group compared with non-GDM controls (0.6 0.08 vs. 0.4 0.03, p < 0.05). Furthermore, the ratio of mitochondrial/nucleic hTERT in CBMCs was correlated with 8-iso-PGF2a levels in cord blood when the two groups of
Please cite this article in press as: Li P, et al. Mitochondrial translocation of human telomerase reverse transcriptase in cord blood mononuclear cells of newborns with gestational diabetes mellitus mothers. Diabetes Res Clin Pract (2014), http://dx.doi.org/10.1016/j.diabres.2013.12.024
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Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.diabres.2013.12.024.
4.4. Neonatal mtDNA content was positively correlated with mitochondrial hTERT in CBMCs
Fig. 2 – The ratio of mitochondrial/nucleic hTERT in CBMCs was increased significantly in the newborns of GDM mothers (n = 26) compared with non-GDM controls (n = 47); *p < 0.001 vs. control. The ratio was calculated as the protein levels of mitochondrial TERT divided by nucleic TERT, which was evaluated in triplicate.
newborns with and without GDM mothers were combined (r = 0.48, p < 0.05) (Fig. 3). These data suggest that there is increased mitochondrial translocation of hTERT in CBMCs under conditions of elevated oxidative stress in GDM newborns.
Neonatal mtDNA content is an indicator of neonatal mitochondrial density and mitochondrial function. Previous studies suggested that mitochondrial hTERT could protect mtDNA from oxidative damage [29,30]. To determine the correlation between mitochondrial hTERT in CBMCs and neonatal mtDNA content, RT-PCR analysis was conducted to assess the relative mtDNA content. Data revealed no significant differences in neonatal mtDNA content between the GDM group and non-GDM controls (median 280.0, IQR 205.5–344.8 vs. median 257.8, IQR 211.4–368.8, respectively) ( p > 0.05), although the mtDNA content in GDM females was reduced compared with non-GDM controls (median 183.8, IQR 148.6–230.0 vs. median 208.7, IQR 174.0–252.4) ( p < 0.05) (Fig. 4). Moreover, the Spearman‘s correlation test showed that the ratio of mitochondrial/nucleic hTERT in CBMCs correlated positively with neonatal mtDNA content in the GDM group (r = 0.6, p < 0.05), non-GDM controls (r = 0.5, p < 0.05), and the two groups combined (r = 0.48, p < 0.05) (Fig. 5). These data suggest a positive relationship between neonatal mtDNA content and mitochondrial hTERT in CBMCs, suggesting that the induction of mitochondrial hTERT in newborns of GDM mothers could protect against oxidative damage to mtDNA. This might help to explain the non-changes in neonatal mtDNA content in GDM pregnancies.
Fig. 3 – Correlation of plasma 8-isoprostaglandin F2a (8-iso-PGF2a, a promising marker for oxidative stress) levels in cord blood with the ratio of mitochondrial/nucleic hTERT in CBMCs when the GDM and control groups combined. The ratio of mitochondrial/nucleic hTERT in CBMCs was elevated significantly with increased levels of 8-iso-PGF2a in cord blood (r = 0.48, p < 0.05). Please cite this article in press as: Li P, et al. Mitochondrial translocation of human telomerase reverse transcriptase in cord blood mononuclear cells of newborns with gestational diabetes mellitus mothers. Diabetes Res Clin Pract (2014), http://dx.doi.org/10.1016/j.diabres.2013.12.024
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Fig. 4 – Changes in the relative mtDNA content (2SDCt) in maternal and cord blood. The change was decreased significantly in the maternal blood in GDM group (median 183.8, IQR 148.6–230.0) compared with control (*p < 0.05). However, there were no significant differences in the mtDNA content of newborns with or without GDM mothers (median 280.0, IQR 205.5–344.8 and median 257.8, IQR 211.4–368.8, respectively) ( p > 0.05).
Fig. 5 – Correlation of neonatal mtDNA content with the ratio of mitochondrial/nucleic hTERT in CBMCs when the GDM and control groups combined. The neonatal mtDNA content was positively correlated with mitochondrial hTERT levels in CBMCs (r = 0.30, p < 0.05). Please cite this article in press as: Li P, et al. Mitochondrial translocation of human telomerase reverse transcriptase in cord blood mononuclear cells of newborns with gestational diabetes mellitus mothers. Diabetes Res Clin Pract (2014), http://dx.doi.org/10.1016/j.diabres.2013.12.024
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5.
Discussion
GDM is a common complication of pregnancy and leads to an adverse intrauterine environment. Although the exposure of a fetus to a GDM intrauterine environment causes predictive adaptive alterations in patterns of fetal metabolism, the protective adaptive responses in utero could predispose infants to develop metabolic disease in later life after environmental conditions improve rapidly [8]. The hypothesis of fetal adaptive programming was proposed long ago, and its underlying mechanisms are complicated and remain elusive [8,37,38]. In recent years, oxidative stress was considered to play an important role, but the mechanism was not fully understood. In the present study, we hypothesized that the mitochondrial translocation of hTERT in CBMCs upon increased oxidative stress could protect the mtDNA of newborns in GDM pregnancies. We observed significantly increased oxidative stress in GDM females and their newborns compared with non-GDM controls, consistent with previous studies [18,19,35]. Mitochondria regulate oxidative stress, and play critical roles in fetal growth and development [24]. MtDNA, as an indicator of mitochondrial function, was protected from oxidative damage by mitochondrial hTERT in both in vitro and in vivo studies [30,39,40]. In this study, hTERT was localized in both the mitochondria and nuclei of CBMCs. Moreover, the ratio of mitochondrial/nucleic hTERT in CBMCs was elevated significantly in the GDM group compared with non-GDM controls, and increased concurrent with oxidative stress in cord blood when the groups of newborns were combined. This suggests increased levels of mitochondrial-localized hTERT in the CBMCs of newborns upon heightened oxidative stress in GDM pregnancies. This is consistent with previous results reporting the accumulation of mitochondrial hTERT after its translocation from nuclei under increased oxidative stress in vitro [27,39]. In support of these results, recent studies identified a mitochondrial-targeting signal (MTS) in the Nterminal sequence of hTERT and two binding regions (ND1 and ND2) in mtDNA genome, which regulate the translocation of TERT from nuclei to mitochondria [30,40]. We found no reduction in the neonatal mtDNA content in the GDM group, although the mtDNA content in GDM females was reduced compared with non-GDM controls, consistent with previous findings [41]. In addition, neonatal mtDNA content was positively correlated with mitochondrial hTERT in CBMCs. This suggests that mitochondrial hTERT in CBMCs could protect mtDNA from oxidative stress in newborns of GDM pregnancies. Evidence for protective effects of mitochondrial TERT on mitochondrial function was reported in cell lines and HUVECs after TERT knockdown, as well as in the hearts of TERT knockout mice [29,30,34]. Therefore, these data suggest that the accumulation of mitochondrial hTERT in CBMCs could protect neonatal mtDNA from oxidative damage in GDM pregnancies. This could be explained by an adaptive response of the fetus to the elevated oxidative stress in the GDM intrauterine environment. An intrauterine environment with heightened oxidative stress was considered to be adverse for fetal growth and development [42–45]. Specifically, mtDNA was important for mitochondrial function and critical for fetal
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development, but was susceptible to the oxidative damage [24,46]. To ensure correct fetal development, increased hTERT was translocated to the mitochondria to protect the neonatal mtDNA under conditions of oxidative stress during GDM pregnancy. This suggests that the mitochondrial translocation of hTERT could be an in utero adaptive response of the fetus to heightened oxidative stress during GDM pregnancies. The most important limitation of this study was the small sample size. This is mainly because a large amount of cord blood was needed for the isolation of CBMCs to extract the total nucleic and mitochondrial proteins due to the low extraction yield. In addition, more mothers had their cord blood collected and stored in a cord blood bank, which prevented us from collecting sufficient blood. To correct for this, we used approximately two controls for each GDM case to reduce the influence of confounding factors. Another limitation was that we used ELISA rather than telomeric repeat amplification protocol (TRAP) to evaluate hTERT protein levels, which may be less accurate. Taken together, the present data provide the first evidence that hTERT was localized in both nuclei and mitochondria in CBMCs, and that the accumulation of mitochondrial hTERT under heightened oxidative stress could protect neonatal mtDNA from oxidative damage during GDM pregnancy. The mitochondrial translocation of hTERT could be an in utero adaptive response of the fetus to elevated oxidative stress. This provides novel insights into the correlation of oxidative stress with mitochondria during fetal development, and may help identify the roles of oxidative stress during fetal programming. However, the short- and long-term consequences of oxidative stress-induced hTERT mitochondrial translocation on the course and outcome of pregnancy remain to be elucidated in larger prospective studies.
Conflict of interest The authors have no competing interests to declare.
Acknowledgements We thank Prof. ZY Zhao and other teammates of Children‘s Hospital of Zhejiang University for providing us with research guidance. We gratefully acknowledge the assistance of Prof. HJ Wan, PI of Development and Disease, as well as Prof. CA Jiang, PI of Developmental and Stem Cell Research Institute of Sichuan University in China, who research the correlation between oxidative stress and mitochondrial function in neurodegeneration.
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Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Mitochondrial translocation of human telomerase reverse transcriptase in cord blood mononuclear cells of newborns with gestational diabetes mellitus mothers§ Ping Li a,b,c, Yu Tong b,c, Huiming Yang a, Shu Zhou e, Fei Xiong a, Tingzhu Huo a, Meng Mao b,c,d,* a Department of Pediatrics, West China Second University Hospital, Sichuan University, No. 17, People‘s South Road, Chengdu 610041, Sichuan Province, PR China b Laboratory of Early Developmental and Injuries, West China Institute of Woman and Children’s Health, West China Second University Hospital, Sichuan University, PR China c Key Laboratory of Obstetric & Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, PR China d Chengdu Women’s and Children’s Central Hospital, No. 1617, Riyue Avenue, Chengdu 610091, Sichuan Province, PR China e Department of Obstetrics, West China Second University Hospital, Sichuan University, No. 17, People‘s South Road, Chengdu 610041, Sichuan Province, PR China
article info
abstract
Article history:
Aims: To better understand the role of oxidative stress in fetal programming, we assessed
Received 23 June 2013
the hypothesis that the mitochondrial translocation of human telomerase reverse tran-
Received in revised form
scriptase (hTERT) could protect neonatal mitochondrial DNA (mtDNA) from oxidative
8 November 2013
damage during pregnancies complicated by gestational diabetes mellitus (GDM).
Accepted 21 December 2013
Methods: 26 GDM mothers and 47 controls and their newborns were enrolled. The plasma
Available online xxx
levels of 8-isoprostaglandin F2a in maternal and cord blood were measured to evaluate
Keywords:
hTERT in cord blood mononuclear cells (CBMCs). Finally, the relative mtDNA content was
oxidative stress. Western blotting was then used to assess the mitochondrial localization of Gestational diabetes
analyzed by real-time PCR.
Fetal programming
Results: GDM mothers and their newborns had significantly higher levels of oxidative stress
Mitochondrial hTERT
than controls. hTERT was localized in both the nuclei and mitochondria of CBMCs, and the
Oxidative stress
increased CBMC mitochondrial hTERT levels were significantly correlated with elevated oxidative stress in newborns. The neonatal mtDNA content in the GDM group was comparable to controls, and was positively correlated with mitochondrial hTERT levels in CBMCs, suggesting that mitochondrial hTERT in CBMCs may have a protective effect on neonatal mtDNA in GDM pregnancies.
§ This study was supported by the national ‘‘11th Five-Year Plan’’ to support science and technology project grants, Ministry of Sciences and Technology, China (2009BAI80B00). * Corresponding author at: Laboratory of Early Developmental and Injuries, West China Institute of Woman and Children’s Health, West China Second University Hospital, Sichuan University, PR China. Tel.: +86 13668171437; fax: +86 02885501698. E-mail address: [email protected] (M. Mao). 0168-8227/$ – see front matter # 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.diabres.2013.12.024
Please cite this article in press as: Li P, et al. Mitochondrial translocation of human telomerase reverse transcriptase in cord blood mononuclear cells of newborns with gestational diabetes mellitus mothers. Diabetes Res Clin Pract (2014), http://dx.doi.org/10.1016/j.diabres.2013.12.024
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Conclusions: This study is the first to suggest that the mitochondrial translocation of hTERT in CBMCs under heightened oxidative stress might protect neonatal mtDNA from oxidative damage in GDM pregnancies. This could be an in utero adaptive response of a fetus that is suffering from elevated oxidative stress, and could help our understanding of the roles of oxidative stress in fetal programming. # 2014 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
Gestational diabetes mellitus (GDM) is a common complication of pregnancy that affects 5–10% of pregnancies in Asian females [1,2]. GDM pregnancy significantly increases the risk of a number of short- and long-term adverse consequences for the fetus, the most significant of which is a predisposition to the development of metabolic syndrome and type 2 diabetes [3–5]. The fetus in a GDM pregnancy is exposed to sustained higher glucose concentrations until birth. To adapt to a GDM intrauterine environment, the pattern of fetal metabolism is altered, including changes in the fetal pancreas allowing the production of increased levels of insulin and epigenetic modifications [6,7]. However, when the environmental conditions improve rapidly after birth, the in utero protective effects of the fetal predictive adaptive responses may have adverse effects on the long-term health of the newborns [8,9]. Although the fetal adaptive programming of metabolic disease has been proposed for a long time, its underlying mechanisms remain elusive. Notably, elevated oxidative stress (OS) is considered to be a major contributor [10–15], since not only GDM females but also their newborns exhibit a heightened level of OS [16–19]. Furthermore, increased OS was also detected in children born during compromised pregnancies, and was potentially related to insulin resistance and obesity in later life [20,21]. OS was reported to alter epigenetic modification via oxidant molecules that act as signaling factors, as well as induce susceptible fetal pancreas dysfunction and subsequent insulin resistance in the offspring [10,15,22]. OS plays an important role in the fetal programming of metabolic disease, although the mechanisms behind these effects remain elusive. A better understanding of the actions of OS in the newborns of GDM mothers is important to identify new ways to reinforce the programming role of OS in metabolic disease. The mitochondrion is a critical organelle that regulates OS, and plays important roles in fetal growth and development [23– 25]. MtDNA is the intrinsic genome of mitochondria, and is responsible for encoding the majority of the respiratory chain enzymes. It is also considered to be an indicator of mitochondrial function [26]. Previous studies demonstrated that mtDNA was protected by mitochondrially localized telomerase reverse transcriptase (TERT) under increased OS. TERT is the catalytic subunit of the telomerase holoenzyme, which is a ribonucleoprotein responsible for the maintenance of telomeres to alleviate replicative senescence and genetic instability. In addition to the telomere-dependent functions of nuclear TERT, studies have suggested that TERT was also localized to the mitochondria [27–30]. Importantly, endogenous mitochondrial TERT is not an artifact of the overexpression of proteins associated with cellular transformation [30–32]. A mitochon-
drial targeting signal (MTS) in the N-terminal sequence of hTERT and two binding regions (ND1 and ND2) in mtDNA were found to play roles in the translocation of TERT from the nucleus to the mitochondria [27,30]. Of note, increased hTERT was exported to the mitochondria and exerted protective effects on mitochondrial function under increased OS [27–30]. In addition, mitochondrial hTERT allowed cancer cells to evade death and multidrug resistance by enhancing mitochondrial function and reducing levels of cellular ROS [33,34]. Collectively, experimental data in vitro and in vivo demonstrated that the increased mitochondrial translocation of hTERT induced by OS plays a putative role in the protection of mitochondrial function. Nevertheless, little information is available on newborns of GDM pregnancies. In this study, we investigated the mitochondrial translocation of hTERT under increased OS in newborns of GDM pregnancies, and assessed its correlation with neonatal mtDNA content. This will help us to better understand the roles of GDM-associated OS in fetal programming.
2.
Materials and methods
2.1.
Recruitment strategy and subjects
Twenty-six pregnant females with GDM (study group) and forty-seven healthy pregnant females without GDM (control group) as well as their newborns were finally included in the study. Pregnant females who underwent in vitro fertilization or with multiple pregnancies, inherited metabolic diseases, pregestational diabetes, and chronic hypertension were excluded. GDM was confirmed using the World Health Organization’s diagnostic criteria. Women who had any of the following were regarded as having GDM: a 1-h 50 g glucose challenge test (GCT) 7.8 mmol/L, fasting plasma glucose of 7.0 mmol/L, 1-h oral glucose tolerance test (75 g OGTT) of 10.0 mmol/L, or 2-h OGTT 8.5 mmol/L. All patients were recruited from the outpatient clinic during their third trimester in the Obstetrical Department of Southwest Second University Hospital affiliated with Sichuan University, China from 2009–2011. All the enrolled females also delivered in this hospital. The Regional and Institutional Committee of Science and Research Ethics of the West China Second University Hospital approved the study, and a written informed consent was obtained from all participants.
2.2.
Baseline data and blood sample collection
Maternal data concerning age, parity, gravidity, height, prepregnancy weight, pregnancy weight gain, family history of
Please cite this article in press as: Li P, et al. Mitochondrial translocation of human telomerase reverse transcriptase in cord blood mononuclear cells of newborns with gestational diabetes mellitus mothers. Diabetes Res Clin Pract (2014), http://dx.doi.org/10.1016/j.diabres.2013.12.024
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diabetes, a history of delivering macrosomia, or low birth weight were collected by a self-administered pregnancy nutrition and background questionnaire. Specialist clinicians collected the birth information of each newborn. Third trimester peripheral blood of pregnant women was drawn and partly centrifuged (3500 g for 10 min at 4 8C) within 30 min of collection. The plasma and blood cells were then frozen in separate aliquots at 80 8C until use. The rest of the whole blood was also stored at 80 8C. In addition, 35 mL venous cord blood was collected from the placental side of the cord after delivery and umbilical cord clamping. Thirty milliliters were used to isolate mononuclear cells (CBMCs) within 24 h, and the rest was processed as described for the maternal peripheral blood.
2.3.
Evaluation of oxidative stress
8-Isoprostaglandin F2a (8-iso-PGF2a), a promising oxidative stress marker, was detected in the plasma of maternal and cord blood using a commercially available Direct 8-iso-PGF2a ELISA kit (Enzo Life Sciences, Farmingdale, USA). A true reflection of both free and esterified isoprostane was measured following the manufacturer’s instructions.
2.4.
Isolation of cord blood mononuclear cells
CBMCs were isolated from the umbilical venous cord blood samples by Ficoll-Hypaque density gradient centrifugation, and stored in liquid nitrogen until analysis.
2.5.
Subcellular fractionation and Western blotting
Mitochondrial fractions were extracted from the CBMCs using the Mitochondria Isolation Kit for cells (Pierce/Thermo, Rockford, USA), and nuclear fractions were extracted with the Nuclear/Cytosol Fractionation Kit (BioVision, Mountain View, USA). The protein concentrations were determined using a Micro-BCA protein concentration determination kit (Pierce/ Thermo). Each type of protein sample was then adjusted to the same concentration and stored in separate aliquots at 80 8C until further analysis. Western blotting was performed to confirm that subcellular fractions were pure and uncontaminated, and to investigate the sub-cellular localization of hTERT. Antibodies used included monoclonal anti-VDAC1/Porin (Abcam, 1:1000; loading control for the mitochondrial fraction), polyclonal anti-Histone H2A.X (Abcam, 1:1000; nuclear fraction) and antihTERT (Abcam, 1:1000).
2.6.
Detection of human telomerase reverse transcriptase
Levels of hTERT in CBMCs were measured using a commercially available hTERT ELISA kit (GenWay Biotech, San Diego, USA). Total mitochondrial and nuclear proteins (1.0 mg/mL) were prepared prior to detection. The ELISA was then performed following the manufacturer’s instructions. The protein levels of hTERT were calculated by subtracting the absorbance value of the negative control from the sample, and normalizing to the positive control.
2.7.
3
Quantification of mtDNA content by real-time PCR
Total DNA was extracted from maternal peripheral and venous cord blood using the QIAamp DNA isolation kit for blood cells (Qiagen, Hilden, Germany). After quantification and adjustment of each genomic DNA concentration using Thermo Scientific Varioskan Flash (Thermo Scientific), samples were stored in separate aliquots at 80 8C until analysis. The copy numbers of mtDNA and nuclear DNA (nDNA) were determined using real-time quantitative polymerase chain reaction (RT-PCR) on a Bio-Rad C1000 thermal cycler equipped with the CFX96TM optical module. MT-RNR2 was selected as a mitochondrial target, and GAPDH as a nuclear target. The nuclear target was used to quantify nuclear DNA and normalize the amount of mtDNA per cell. The primer sequences MT-RNR2 forward 50 -CCAAACCCACTCCACCTTAC30 , and reverse 50 -TCATCTTTCCCTTGCGGTA-30 ; and GAPDH forward 50 -TTCAACAGCGACACCCACT-30 , and reverse 50 CCAGCCACATACCAGGAAAT-30 were reported previously by Pejznochova et al. [25]. Real-time PCR amplification was performed in 10 mL containing 2 SsoFastTM EvaGreen1 Supermix (Bio-Rad), 200 nM each primer, and 0.5 mL of each analyzed DNA sample at a concentration of 50 ng/mL. The PCR conditions were as follows: one cycle of 95 8C for 15 min, 40 cycles of 95 8C for 15 s and 60.6 8C for 30 s (MT-RNR2) or 60.8 8C for 15 s (GAPDH), and 72 8C for 30 s, followed by 72 8C for 5 min and a melting curve from 72 to 92 8C (with fluorescence readings every 1 8C/10 s). Two-fold serial dilutions of the genomic DNA (from 100 to 6.25 ng) were included in each run to generate the calibration curve. All samples were run in triplicate for each gene. The standard deviation of the mtDNA assay in triplicate repeats of different samples was 8%. Data were analyzed using the comparative Ct method, where Ct was the cycle number at which the instrument first detected fluorescence above background noise. The D cycle threshold (DCt) values of each sample was obtained by subtracting the values for the reference gene from the sample Ct, thus normalizing to nuclear DNA. For each experimental sample, the 2DCt was calculated, and data are presented as relative quantification. The slope of the calibration curve reflected the reaction efficiency, and each correlation coefficient was between 0.99 and 1.00. Melting curve analysis and electrophoresis were used to analyze reaction specificity.
3.
Statistical analysis
The SPSS1 statistical package, version 13.0 (SPSS, Inc., Chicago, Illinois) was used for statistical analysis. Quantitative data with normal distribution were expressed as the mean standard deviation (SD), and analyzed by the independent sample t-test. Non-normal quantitative data were expressed as the medians with interquartile ranges (IQR), and analyzed using Mann–Whitney U-test. The correlation between two variables was performed by Pearson correlation test or Spearman rank correlation test according to the data distribution. A two-tailed p < 0.05 was considered to be statistically significant.
Please cite this article in press as: Li P, et al. Mitochondrial translocation of human telomerase reverse transcriptase in cord blood mononuclear cells of newborns with gestational diabetes mellitus mothers. Diabetes Res Clin Pract (2014), http://dx.doi.org/10.1016/j.diabres.2013.12.024
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Table 1 – Clinical characteristic of enrolled mothers and their newborns.
Maternal data Age (years) Parity Gravidity Prepregnancy BMI (kg/m2) Weight gain (kg) Fasting plasma glucose (mmol/L) Family history of diabetes (%) Macrosomia history (%) Low birth weight history (%) Neonate data Gestational age at delivery (wks) Birth weight (g) Birth weight ratio (%) Microsomia (%) Male ratio (%) Cesarean section (%) Fasting plasma glucose (mmol/L)
GDM (n = 26)
Control (n = 47)
31.8 (29.8–37.4) 1.12 0.3 2.4 1.5 21.6 3.2 13.8 3.9 5.1 0.5 38.5 26.9 11.4
30.2 (28.8–32.2) 1.11 0.3 1.8 1.2 20.3 2.1 13.2 3.2 4.4 0.5 17.0 6.4 6.4
Ns Ns 0.00 0.04 Ns 0.00 0.01 0.04 Ns
38.7 1.2 3383.7 542.7 1.1 0.1 1.5 61.5 34.6 4.1 (1.7–4.5)
38.6 1.0 3347.8 370.4 1.0 0.2 2.1 42.6 28.9 3.3(2.9–61.9)
Ns Ns Ns Ns Ns Ns Ns
P
Note: Values are medians with interquartile ranges or mean SDs. Ns, no significance.
4.
Results
4.1.
Clinical characteristics
Twenty-six GDM mothers and forty-seven non-GDM controls and their newborns were enrolled in the study. The GDM and control groups were well matched for gestational age. The clinical characteristics of the mothers and newborns are shown in Table 1. The median age of GDM mothers was 31.8 years, which was slightly (but not significantly) older than non-GDM mothers (30.2 years). Although GDM females had higher gravidity than the controls, parity was similar between groups. In addition, GDM women had a higher pre-pregnancy BMI, but weight gain was similar in both groups. Fasting plasma glucose levels were significantly higher in GDM
mothers compared with control. A family history of diabetes and history of delivering macrosomia were also more frequent in the GDM group. However, no significant differences were found in birth weights of newborns in the two groups.
4.2. Higher levels of oxidative stress in GDM mothers and their newborns To evaluate oxidative stress in GDM mothers and their newborns, an ELISA kit was used to measure the plasma levels of 8-iso-PGF2a, a stable and specific marker of oxidative stress. Compared with controls, plasma levels of 8-iso-PGF2a were increased significantly in both the maternal blood of GDM females (9336.3 2919.9 vs. 7390.9 2211.1, p < 0.05) and in the cord blood of their newborns (5792.4 1088.6 vs. 4430.6 908.0, p < 0.05; Fig. 1). Consistent with previous studies [18,19,35], our data demonstrated that both GDM mothers and their newborns were suffering from increased oxidative stress compared with non-GDM controls.
4.3. Mitochondrial hTERT in CBMCs positively correlated with oxidative stress in cord blood
Fig. 1 – Plasma levels of 8-isoprostaglandin F2a (8-isoPGF2a), a stable and specific marker for oxidative stress, were increased significantly in both GDM women and their newborns compared with non-GDM controls; *p < 0.001 vs. control. Twenty-six separate GDM maternal and cord blood samples, and forty-seven separate control maternal and cord blood samples were measured in triplicate.
Previous studies reported endogenous mitochondrial hTERT, and oxidative stress could induce accumulation of mitochondrial hTERT by its translocation from the nucleus [29,31,36]. Therefore, we used western blotting to determine whether mitochondrial hTERT could be detected in CBMCs. Data revealed that hTERT was localized in both the mitochondria and nuclei of CBMCs (Supplementary Fig. 1). Next, ELISAs were performed to determine whether there were different levels of mitochondrial hTERT in CBMCs from newborns with and without GDM mothers. As shown in Fig. 2, the ratio of mitochondrial/nucleic hTERT in CBMCs was elevated significantly in the GDM group compared with non-GDM controls (0.6 0.08 vs. 0.4 0.03, p < 0.05). Furthermore, the ratio of mitochondrial/nucleic hTERT in CBMCs was correlated with 8-iso-PGF2a levels in cord blood when the two groups of
Please cite this article in press as: Li P, et al. Mitochondrial translocation of human telomerase reverse transcriptase in cord blood mononuclear cells of newborns with gestational diabetes mellitus mothers. Diabetes Res Clin Pract (2014), http://dx.doi.org/10.1016/j.diabres.2013.12.024
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Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.diabres.2013.12.024.
4.4. Neonatal mtDNA content was positively correlated with mitochondrial hTERT in CBMCs
Fig. 2 – The ratio of mitochondrial/nucleic hTERT in CBMCs was increased significantly in the newborns of GDM mothers (n = 26) compared with non-GDM controls (n = 47); *p < 0.001 vs. control. The ratio was calculated as the protein levels of mitochondrial TERT divided by nucleic TERT, which was evaluated in triplicate.
newborns with and without GDM mothers were combined (r = 0.48, p < 0.05) (Fig. 3). These data suggest that there is increased mitochondrial translocation of hTERT in CBMCs under conditions of elevated oxidative stress in GDM newborns.
Neonatal mtDNA content is an indicator of neonatal mitochondrial density and mitochondrial function. Previous studies suggested that mitochondrial hTERT could protect mtDNA from oxidative damage [29,30]. To determine the correlation between mitochondrial hTERT in CBMCs and neonatal mtDNA content, RT-PCR analysis was conducted to assess the relative mtDNA content. Data revealed no significant differences in neonatal mtDNA content between the GDM group and non-GDM controls (median 280.0, IQR 205.5–344.8 vs. median 257.8, IQR 211.4–368.8, respectively) ( p > 0.05), although the mtDNA content in GDM females was reduced compared with non-GDM controls (median 183.8, IQR 148.6–230.0 vs. median 208.7, IQR 174.0–252.4) ( p < 0.05) (Fig. 4). Moreover, the Spearman‘s correlation test showed that the ratio of mitochondrial/nucleic hTERT in CBMCs correlated positively with neonatal mtDNA content in the GDM group (r = 0.6, p < 0.05), non-GDM controls (r = 0.5, p < 0.05), and the two groups combined (r = 0.48, p < 0.05) (Fig. 5). These data suggest a positive relationship between neonatal mtDNA content and mitochondrial hTERT in CBMCs, suggesting that the induction of mitochondrial hTERT in newborns of GDM mothers could protect against oxidative damage to mtDNA. This might help to explain the non-changes in neonatal mtDNA content in GDM pregnancies.
Fig. 3 – Correlation of plasma 8-isoprostaglandin F2a (8-iso-PGF2a, a promising marker for oxidative stress) levels in cord blood with the ratio of mitochondrial/nucleic hTERT in CBMCs when the GDM and control groups combined. The ratio of mitochondrial/nucleic hTERT in CBMCs was elevated significantly with increased levels of 8-iso-PGF2a in cord blood (r = 0.48, p < 0.05). Please cite this article in press as: Li P, et al. Mitochondrial translocation of human telomerase reverse transcriptase in cord blood mononuclear cells of newborns with gestational diabetes mellitus mothers. Diabetes Res Clin Pract (2014), http://dx.doi.org/10.1016/j.diabres.2013.12.024
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Fig. 4 – Changes in the relative mtDNA content (2SDCt) in maternal and cord blood. The change was decreased significantly in the maternal blood in GDM group (median 183.8, IQR 148.6–230.0) compared with control (*p < 0.05). However, there were no significant differences in the mtDNA content of newborns with or without GDM mothers (median 280.0, IQR 205.5–344.8 and median 257.8, IQR 211.4–368.8, respectively) ( p > 0.05).
Fig. 5 – Correlation of neonatal mtDNA content with the ratio of mitochondrial/nucleic hTERT in CBMCs when the GDM and control groups combined. The neonatal mtDNA content was positively correlated with mitochondrial hTERT levels in CBMCs (r = 0.30, p < 0.05). Please cite this article in press as: Li P, et al. Mitochondrial translocation of human telomerase reverse transcriptase in cord blood mononuclear cells of newborns with gestational diabetes mellitus mothers. Diabetes Res Clin Pract (2014), http://dx.doi.org/10.1016/j.diabres.2013.12.024
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5.
Discussion
GDM is a common complication of pregnancy and leads to an adverse intrauterine environment. Although the exposure of a fetus to a GDM intrauterine environment causes predictive adaptive alterations in patterns of fetal metabolism, the protective adaptive responses in utero could predispose infants to develop metabolic disease in later life after environmental conditions improve rapidly [8]. The hypothesis of fetal adaptive programming was proposed long ago, and its underlying mechanisms are complicated and remain elusive [8,37,38]. In recent years, oxidative stress was considered to play an important role, but the mechanism was not fully understood. In the present study, we hypothesized that the mitochondrial translocation of hTERT in CBMCs upon increased oxidative stress could protect the mtDNA of newborns in GDM pregnancies. We observed significantly increased oxidative stress in GDM females and their newborns compared with non-GDM controls, consistent with previous studies [18,19,35]. Mitochondria regulate oxidative stress, and play critical roles in fetal growth and development [24]. MtDNA, as an indicator of mitochondrial function, was protected from oxidative damage by mitochondrial hTERT in both in vitro and in vivo studies [30,39,40]. In this study, hTERT was localized in both the mitochondria and nuclei of CBMCs. Moreover, the ratio of mitochondrial/nucleic hTERT in CBMCs was elevated significantly in the GDM group compared with non-GDM controls, and increased concurrent with oxidative stress in cord blood when the groups of newborns were combined. This suggests increased levels of mitochondrial-localized hTERT in the CBMCs of newborns upon heightened oxidative stress in GDM pregnancies. This is consistent with previous results reporting the accumulation of mitochondrial hTERT after its translocation from nuclei under increased oxidative stress in vitro [27,39]. In support of these results, recent studies identified a mitochondrial-targeting signal (MTS) in the Nterminal sequence of hTERT and two binding regions (ND1 and ND2) in mtDNA genome, which regulate the translocation of TERT from nuclei to mitochondria [30,40]. We found no reduction in the neonatal mtDNA content in the GDM group, although the mtDNA content in GDM females was reduced compared with non-GDM controls, consistent with previous findings [41]. In addition, neonatal mtDNA content was positively correlated with mitochondrial hTERT in CBMCs. This suggests that mitochondrial hTERT in CBMCs could protect mtDNA from oxidative stress in newborns of GDM pregnancies. Evidence for protective effects of mitochondrial TERT on mitochondrial function was reported in cell lines and HUVECs after TERT knockdown, as well as in the hearts of TERT knockout mice [29,30,34]. Therefore, these data suggest that the accumulation of mitochondrial hTERT in CBMCs could protect neonatal mtDNA from oxidative damage in GDM pregnancies. This could be explained by an adaptive response of the fetus to the elevated oxidative stress in the GDM intrauterine environment. An intrauterine environment with heightened oxidative stress was considered to be adverse for fetal growth and development [42–45]. Specifically, mtDNA was important for mitochondrial function and critical for fetal
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development, but was susceptible to the oxidative damage [24,46]. To ensure correct fetal development, increased hTERT was translocated to the mitochondria to protect the neonatal mtDNA under conditions of oxidative stress during GDM pregnancy. This suggests that the mitochondrial translocation of hTERT could be an in utero adaptive response of the fetus to heightened oxidative stress during GDM pregnancies. The most important limitation of this study was the small sample size. This is mainly because a large amount of cord blood was needed for the isolation of CBMCs to extract the total nucleic and mitochondrial proteins due to the low extraction yield. In addition, more mothers had their cord blood collected and stored in a cord blood bank, which prevented us from collecting sufficient blood. To correct for this, we used approximately two controls for each GDM case to reduce the influence of confounding factors. Another limitation was that we used ELISA rather than telomeric repeat amplification protocol (TRAP) to evaluate hTERT protein levels, which may be less accurate. Taken together, the present data provide the first evidence that hTERT was localized in both nuclei and mitochondria in CBMCs, and that the accumulation of mitochondrial hTERT under heightened oxidative stress could protect neonatal mtDNA from oxidative damage during GDM pregnancy. The mitochondrial translocation of hTERT could be an in utero adaptive response of the fetus to elevated oxidative stress. This provides novel insights into the correlation of oxidative stress with mitochondria during fetal development, and may help identify the roles of oxidative stress during fetal programming. However, the short- and long-term consequences of oxidative stress-induced hTERT mitochondrial translocation on the course and outcome of pregnancy remain to be elucidated in larger prospective studies.
Conflict of interest The authors have no competing interests to declare.
Acknowledgements We thank Prof. ZY Zhao and other teammates of Children‘s Hospital of Zhejiang University for providing us with research guidance. We gratefully acknowledge the assistance of Prof. HJ Wan, PI of Development and Disease, as well as Prof. CA Jiang, PI of Developmental and Stem Cell Research Institute of Sichuan University in China, who research the correlation between oxidative stress and mitochondrial function in neurodegeneration.
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