Maria Schindler

Maternal Diabetes Impairs Gastrulation and Insulin and IGF-I Receptor Expression in Rabbit Blastocysts

Women with type 1 diabetes are subfertile. Diabetes negatively affects pregnancy by causing early miscarriage and poor prenatal outcomes. In this study we examine consequences of maternal type 1 diabetes on early embryo development, metabolic gene expression, and the pattern of insulin receptor (IR) and IGF-I receptor (IGF-IR) distribution in rabbit blastocysts. In female rabbits, type 1 diabetes was induced by alloxan treatment. Six-day-old blastocysts were recovered and assessed for receptor distribution and metabolic gene expression. In vitro culture of blastocysts was performed in medium containing 1 mm, 10 mm, or 25 mm glucose, simulating normo- and hyperglycemic developmental condition in vitro. The fertility rate of the diabetic rabbits clearly mirrored subfertility with a drop in blastocyst numbers by 40% (13.3 blastocysts in diabetic vs. 21.9 in control females). In blastocysts onset and progression of gastrulation was delayed and expression of IR and IGF-IR and their metabolic target genes (hexokinase, phosphoenolpyruvate carboxykinase), both in vivo and in vitro, was down-regulated. The amount of apoptotic cells in the embryonic disc was increased, correlating closely with the reduced transcription of the bcl-x(L) gene. Blastocyst development is clearly impaired by type 1 diabetes during early pregnancy. Insulin-stimulated metabolic genes and IR and IGF-IR are down-regulated, resulting in reduced insulin and IGF sensitivity and a delay in development. Dysregulation of the IGF system and embryonic glucose metabolism are potential reasons for diabetogenous subfertility and embryopathies and start as soon as during the first days of life.

Insulin growth factor adjustment in preimplantation rabbit blastocysts and uterine tissues in response to maternal type 1 diabetes

Insulin-like growth factors (IGFs) are well-known regulators of embryonic growth and differentiation. IGF function is closely related to insulin action. IGFs are available to the preimplantation embryo through maternal blood (endocrine action), uterine secretions (paracrine action) and by the embryo itself (autocrine action). In rabbit blastocysts, embryonic IGF1 and IGF2 are specifically strong in the embryoblast (ICM). Signalling of IGFs and insulin in blastocysts follows the classical pathway with Erk1/2 and Akt kinase activation. The aim of this study was to analyse signalling of IGFs in experimental insulin dependent diabetes (exp IDD) in pregnancy, employing a diabetic rabbit model with uterine hypoinsulinemia and hyperglycaemia. Exp IDD was induced in female rabbits by alloxan treatment prior to mating. At 6 days p.c., the maternal and embryonic IGFs were quantified by RT-PCR and ELISA. In pregnant females, hepatic IGF1 expression and IGF1 serum levels were decreased while IGF1 and IGF2 were increased in endometrium. In blastocysts, IGF1 RNA and protein was approx. 7.5-fold and 2-fold higher, respectively, than in controls from normoglycemic females. In cultured control blastocysts supplemented with IGF1 or insulin in vitro for 1 or 12 h, IGF1 and insulin receptors as well as IGF1 and IGF2 were downregulated. In cultured T1D blastocysts activation of Akt and Erk1/2 was impaired with lower amounts of total Akt and Erk1/2 protein and a reduced phosphorylation capacity after IGF1 supplementation. Our data show that the IGF axis is severely altered in embryo-maternal interactions in exp IDD pregnancy. Both, the endometrium and the blastocyst produce more IGF1 and IGF2. The increased endogenous IGF1 and IGF2 expression by the blastocyst compensates for the loss of systemic insulin and IGF. However, this counterbalance does not fill the gap of the reduced insulin/IGF sensitivity, leading to a developmental delay of blastocysts in exp IDD pregnancy.

Accumulation of advanced glycation end products in the rabbit blastocyst under maternal diabetes

Diabetes mellitus (DM) during pregnancy is one of the leading causes of perinatal morbidity and birth defects. The mechanism by which maternal hyperglycemia, the major teratogenic factor, induces embryonic malformations remains unclear. Advanced glycation end products (AGEs) are known to accumulate during the course of DM and contribute to the development of diabetic complications. Employing a diabetic rabbit model, we investigated the influence of maternal hyperglycemia during the preimplantation period on AGE formation (pentosidine, argpyrimidine, and N(ϵ)-carboxymethyllysine (CML)) in the reproductive tract and the embryo itself. As a consequence of type 1 DM, the AGE levels in blood plasma increased up to 50%, correlating closely with an AGE accumulation in the endometrium of diabetic females. Embryos from diabetic mothers had increased protein-bound CML levels and showed enhanced fluorescent signals for AGE-specific fluorescence in the blastocyst cavity fluid (BCF). The quantification of CML by HPLC-mass spectrometry (MS/MS) showed a higher amount of soluble CML in the BCF of blastocysts from diabetic rabbits (0.26±0.05 μmol/l) compared with controls (0.18±0.02 μmol/l). The high amount of AGEs in blastocysts from diabetic mothers correlates positively with an increased AGER (receptor for AGE (RAGE)) mRNA expression. Our study gives alarming insights into the consequences of poorly controlled maternal diabetes for AGE formation in the embryo. Maternal hyperglycemia during the preimplantation period is correlated with an increase in AGE formation in the uterine environment and the embryo itself. This may influence the development of the embryo through increased AGE-mediated cellular stress by RAGEs.

cAMP-Responsive Element Binding Protein: A Vital Link in Embryonic Hormonal Adaptation

The transcription factor cAMP responsive element-binding protein (CREB) and activating transcription factors (ATFs) are downstream components of the insulin/IGF cascade, playing crucial roles in maintaining cell viability and embryo survival. One of the CREB target genes is adiponectin, which acts synergistically with insulin. We have studied the CREB-ATF-adiponectin network in rabbit preimplantation development in vivo and in vitro. From the blastocyst stage onwards, CREB and ATF1, ATF3, and ATF4 are present with increasing expression for CREB, ATF1, and ATF3 during gastrulation and with a dominant expression in the embryoblast (EB). In vitro stimulation with insulin and IGF-I reduced CREB and ATF1 transcripts by approximately 50%, whereas CREB phosphorylation was increased. Activation of CREB was accompanied by subsequent reduction in adiponectin and adiponectin receptor (adipoR)1 expression. Under in vivo conditions of diabetes type 1, maternal adiponectin levels were up-regulated in serum and endometrium. Embryonic CREB expression was altered in a cell lineage-specific pattern. Although in EB cells CREB localization did not change, it was translocated from the nucleus into the cytosol in trophoblast (TB) cells. In TB, adiponectin expression was increased (diabetic 427.8 ± 59.3 pg/mL vs normoinsulinaemic 143.9 ± 26.5 pg/mL), whereas it was no longer measureable in the EB. Analysis of embryonic adipoRs showed an increased expression of adipoR1 and no changes in adipoR2 transcription. We conclude that the transcription factors CREB and ATFs vitally participate in embryo-maternal cross talk before implantation in a cell lineage-specific manner. Embryonic CREB/ATFs act as insulin/IGF sensors. Lack of insulin is compensated by a CREB-mediated adiponectin expression, which may maintain glucose uptake in blastocysts grown in diabetic mothers.

Maternal diabetes promotes mTORC1 downstream signalling in rabbit preimplantation embryos

The mammalian target of rapamycin complex 1 (mTORC1) is known to be a central cellular nutrient sensor and master regulator of protein metabolism; therefore, it is indispensable for normal embryonic development. We showed previously in a diabetic pregnancy that embryonic mTORC1 phosphorylation is increased in case of maternal hyperglycaemia and hypoinsulinaemia. Further, the preimplantation embryo is exposed to increased L-leucine levels during a diabetic pregnancy. To understand how mTOR signalling is regulated in preimplantation embryos, we examined consequences of L-leucine and glucose stimulation on mTORC1 signalling and downstream targets in in vitro cultured preimplantation rabbit blastocysts and in vivo. High levels of L-leucine and glucose lead to higher phosphorylation of mTORC1 and its downstream target ribosomal S6 kinase 1 (S6K1) in these embryos. Further, L-leucine supplementation resulted in higher embryonic expression of genes involved in cell cycle (cyclin D1; CCND1), translation initiation (eukaryotic translation initiation factor 4E; EIF4E), amino acid transport (large neutral amino acid transporter 2; Lat2: gene SLC7A8) and proliferation (proliferating cell nuclear antigen; PCNA) in a mTORC1-dependent manner. Phosphorylation of S6K1 and expression patterns of CCND1 and EIF4E were increased in embryos from diabetic rabbits, while the expression of proliferation marker PCNA was decreased. In these embryos, protein synthesis was increased and autophagic activity was decreased. We conclude that mammalian preimplantation embryos sense changes in nutrient supply via mTORC1 signalling. Therefore, mTORC1 may be a decisive mediator of metabolic programming in a diabetic pregnancy.

Maternal Diabetes Leads to Adaptation in Embryonic Amino Acid Metabolism during Early Pregnancy

During pregnancy an adequate amino acid supply is essential for embryo development and fetal growth. We have studied amino acid composition and branched chain amino acid (BCAA) metabolism at day 6 p.c. in diabetic rabbits and blastocysts. In the plasma of diabetic rabbits the concentrations of 12 amino acids were altered in comparison to the controls. Notably, the concentrations of the BCAA leucine, isoleucine and valine were approximately three-fold higher in diabetic rabbits than in the control. In the cavity fluid of blastocysts from diabetic rabbits BCAA concentrations were twice as high as those from controls, indicating a close link between maternal diabetes and embryonic BCAA metabolism. The expression of BCAA oxidizing enzymes and BCAA transporter was analysed in maternal tissues and in blastocysts. The RNA amounts of three oxidizing enzymes, i.e. branched chain aminotransferase 2 (Bcat2), branched chain ketoacid dehydrogenase (Bckdha) and dehydrolipoyl dehydrogenase (Dld), were markedly increased in maternal adipose tissue and decreased in liver and skeletal muscle of diabetic rabbits than in those of controls. Blastocysts of diabetic rabbits revealed a higher Bcat2 mRNA and protein abundance in comparison to control blastocysts. The expression of BCAA transporter LAT1 and LAT2 were unaltered in endometrium of diabetic and healthy rabbits, whereas LAT2 transcripts were increased in blastocysts of diabetic rabbits. In correlation to high embryonic BCAA levels the phosphorylation amount of the nutrient sensor mammalian target of rapamycin (mTOR) was enhanced in blastocysts caused by maternal diabetes. These results demonstrate a direct impact of maternal diabetes on BCAA concentrations and degradation in mammalian blastocysts with influence on embryonic mTOR signalling.

Adiponectin stimulates lipid metabolism via AMPK in rabbit blastocysts

Abstract STUDY QUESTION How does a maternal diabetic hyperadiponectineamia affect signal transduction and lipid metabolism in rabbit preimplantation blastocysts? SUMMARY ANSWER In a diabetic pregnancy increased levels of adiponectin led to a switch in embryonic metabolism towards a fatty acid-dependent energy metabolism, mainly affecting genes that are responsible for fatty acid uptake and turnover. WHAT IS KNOWN ALREADY Although studies in cell culture experiments have shown that adiponectin is able to regulate lipid metabolism via 5′-AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor α (PPARα), data on the effects of adiponectin on embryonic lipid metabolism are not available. In a diabetic pregnancy in rabbits, maternal adiponectin levels are elevated fourfold and are accompanied by an increase in intracellular lipid droplets in blastocysts, implying consequences for the embryonic hormonal and metabolic environment. STUDY DESIGN, SIZE, DURATION Rabbit blastocysts were cultured in vitro with adiponectin (1 μg/ml) and with the specific AMPK-inhibitor Compound C for 15 min, 1 h and 4 h (N ≥ 3 independent experiments: for RNA analysis, n ≥ 4 blastocysts per treatment group; for protein analysis three blastocysts pooled per sample and three samples used per experiment). Adiponectin signalling was verified in blastocysts grown in vivo from diabetic rabbits with a hyperadiponectinaemia (N ≥ 3 independent experiments, n ≥ 4 samples per treatment group, eight blastocysts pooled per sample). PARTICIPANTS/MATERIALS, SETTING, METHODS In these blastocysts, expression of molecules involved in adiponectin signalling [adaptor protein 1 (APPL1), AMPK, acetyl-CoA carboxylase (ACC), p38 mitogen-activated protein kinases (p38 MAPK)], lipid metabolism [PPARα, cluster of differentiation 36 (CD36), fatty acid transport protein 4 (FATP4), fatty acid binding protein (FABP4), carnitine palmityl transferase 1 (CPT1), hormone-senstive lipase (HSL), lipoprotein lipase (LPL)] and members of the insulin/insulin-like growth factor (IGF)-system [IGF1, IGF2, insulin receptor (InsR), IGF1 receptor (IGF1R)] were analyzed by quantitative RT-PCR and western blot. Analyses were performed in both models, i.e. adiponectin stimulated blastocysts (in vitro) and in blastocysts grown in vivo under increased adiponectin levels caused by a maternal diabetes mellitus. MAIN RESULTS AND THE ROLE OF CHANCE In both in vitro and in vivo models adiponectin increased AMPK and ACC phosphorylation, followed by an activation of the transcription factor PPARα, and CPT1, the key enzyme of β-oxidation (all P < 0.05 versus control). Moreover, mRNA levels of the fatty acid transporters CD36, FATP4 and FABP4, and HSL were upregulated by adiponectin/AMPK signalling (all P < 0.05 versus control). Under diabetic developmental conditions the amount of p38 MAPK was upregulated (P < 0.01 versus non-diabetic), which was not observed in blastocysts cultured in vitro with adiponectin, indicating that the elevated p38 MAPK was not related to adiponectin. However, a second effect of adiponectin has to be noted: its intensification of insulin sensitivity, by regulating IGF availability and InsR/IGF1R expression. LARGE SCALE DATA Not applicable. LIMITATIONS REASONS FOR CAUTION There are two main limitations for our study. First, human and rabbit embryogenesis can only be compared during blastocyst development. Therefore, the inferences from our findings are limited to the embryonic stages investigated here. Second, the increased adiponectin levels and lack of maternal insulin is only typical for a diabetes mellitus type one model. WIDER IMPLICATIONS OF THE FINDINGS This is the first mechanistic study demonstrating a direct influence of adiponectin on lipid metabolism in preimplantation embryos. The numbers of young women with a diabetes mellitus type one are increasing steadily. We have shown that preimplantation embryos are able to adapt to changes in the uterine milieu, which is mediated by the adiponectin/AMPK signalling. A tightly hormonal control during pregnancy is essential for survival and proper development. In this control process, adiponectin plays a more important role than known so far. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the German Research Council (DFG RTG ProMoAge 2155), the EU (FP7 Epihealth No. 278418, FP7-EpiHealthNet N°317146), COST Action EpiConcept FA 1201 and SALAAM BM 1308. The authors have no conflict(s) of interest to disclose.

A Diabetic Pregnancy Alters the Expression of Stress-Related Receptors in Gastrulating Rabbit Blastocyst and in the Reproductive Tract.

The incidence of diabetes mellitus for young people rises since years. A preconceptional diabetes mellitus leads to subfertility. Most of the causes for a diabetic subfertility are still unknown. Stress can significantly deteriorate glycemic control in diabetes. Several mechanisms by which “stress hormones”, like adrenaline and cortisol or corticosterone, can contribute to the regulation of glucose homeostasis have been identified. Using reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR, we examined the expression of adrenergic receptors and the glucocorticoid receptor transcripts in the female rabbit reproductive tract and in gastrulating blastocysts developed in normoinsulinemic mothers and in mothers with experimentally induced diabetes mellitus type 1. The glucocorticoid receptor expression was detected in the reproductive tract as well as in gastrulating blastocysts at a high level. In maternal endometrium, α1D-, α2A-, β1-, and β2-adrenergic receptors were expressed, whereby β1 transcript was not detectable in the endometrium from diabetic mothers. In preimplantation embryos, all 9 adrenergic receptors were expressed, most of them predominantly in the embryoblast. A maternal diabetes mellitus altered α2A-adrenergic receptor expression in the blastocyst and reversed the ratio of α2A transcript quantity between embryoblast and trophoblast. Our results show that the maternal reproductive tract and the preimplantation embryo express a distinct pattern of the stress response system. Alterations in the pattern and/or in functionality are likely linked to subfertility in diabetes mellitus.

A short periconceptional exposure to maternal type-1 diabetes is sufficient to disrupt the feto-placental phenotype in a rabbit model

Tight metabolic control of type-1 diabetes is essential during gestation, but it could be crucial during the periconception period. Feto-placental consequences of maternal type-1 diabetes around the time of conception need to be explored. Using a rabbit model, type-1 diabetes was induced by alloxan 7 days before mating. Glycemia was maintained at 15-20 mmol/L with exogenous insulin injections to prevent ketoacidosis. At 4 days post-conception (dpc), embryos were collected from diabetic (D) or normoglycemic control (C) dams, respectively, and transferred into non-diabetic recipients. At 28dpc, D- and C-feto-placental units were collected for biometry, placental analyses and lipid profiles. D-fetuses were growth-retarded, hyperglycemic and dyslipidemic compared to C-fetuses. The efficiency of D-placentas was associated with an increased gene expression related to nutrient supply and lipid metabolism whereas volume density of fetal vessels decreased. Fetal plasma, placental and fetal liver membranes had specific fatty acid signatures depending on embryonic origin. Tissues from D-fetuses contained more omega-6 polyunsaturated fatty acids. The concentrations of docosahexaenoic acid decreased while linoleic acid increased in the heart of D-fetuses. This study demonstrates that a short exposure to maternal type-1 diabetes in the periconception window, until the blastocyst stage, is able to irreversibly malprogram the feto-placental phenotype, through precocious and persistent structural and molecular adaptations of placenta.

Cholesterol metabolism in rabbit blastocysts under maternal diabetes

In the rabbit reproductive model, maternal experimentally induced insulin-dependent diabetes mellitus (expIDD) leads to accumulation of lipid droplets in blastocysts. Cholesterol metabolism is a likely candidate to explain such metabolic changes. Therefore, in the present study we analysed maternal and embryonic cholesterol concentrations and expression of related genes in vivo (diabetic model) and in vitro (embryo culture in hyperglycaemic medium). In pregnant expIDD rabbits, the serum composition of lipoprotein subfractions was changed, with a decrease in high-density lipoprotein cholesterol and an increase in very low-density lipoprotein cholesterol; in uterine fluid, total cholesterol concentrations were elevated. Expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), very low-density lipoprotein receptor (VLDLR), sterol regulatory element binding transcription factor 2 (SREBF2), insulin-induced gene-1 (INSIG1) and cholesterol 7α-hydroxylase (CYP7A1) mRNA was decreased in the liver and low-density lipoprotein receptor (LDLR) mRNA expression was decreased in the adipose tissue of diabetic rabbits. In embryos from diabetic rabbits, the mean (±s.e.m.) ratio of cholesterol concentrations in trophoblasts to embryoblasts was changed from 1.27±2.34 (control) to 0.88±3.85 (expIDD). Rabbit blastocysts expressed HMGCR, LDLR, VLDLR, SREBF2 and INSIG1 but not CYP7A1, without any impairment of expression as a result of maternal diabetes. In vitro hyperglycaemia decreased embryonic HMGCR and SREBF2 transcription in rabbit blastocysts. The findings of the present study show that a diabetic pregnancy leads to distinct changes in maternal cholesterol metabolism with a minor effect on embryo cholesterol metabolism.