Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity.

Late-onset Alzheimer's disease (AD) is a complex multifactorial disease. The greatest known risk factor for late-onset AD is the E4 allele of the apolipoprotein E (APOE), while increasing age is the greatest known non-genetic risk factor. The cell type-specific functions of neural stem cells (NSCs), in particular their stem cell plasticity, remain poorly explored in the context of AD pathology. Here, we describe a new model that employs late-onset AD patient-derived induced pluripotent stem cells (iPSCs) to generate NSCs and to examine the role played by APOE4 in the expression of aging markers such as sirtuin 1 (SIRT1) in comparison to healthy subjects carrying APOE3. The effect of aging was investigated by using iPSC-derived NSCs from old age subjects as healthy matched controls. Transcript and protein analysis revealed that genes were expressed differently in NSCs from late-onset AD patients, e.g., exhibiting reduced autophagy-related protein 7 (ATG7), phosphatase and tensin homolog (PTEN), and fibroblast growth factor 2 (FGF2). Since SIRT1 expression differed between APOE3 and APOE4 NSCs, the suppression of APOE function in NSCs also repressed the expression of SIRT1. However, the forced expression of APOE3 by plasmids did not recover differently expressed genes. The altered aging markers indicate decreased plasticity of NSCs. Our study provides a suitable in vitro model to investigate changes in human NSCs associated with aging, APOE4, and late-onset AD.

Advanced maternal age leads to changes within the insulin/IGF system and lipid metabolism in the reproductive tract and preimplantation embryo: insights from the rabbit model.

Reproductive potential in women declines with age. The impact of ageing on embryo-maternal interactions is still unclear. Rabbits were used as a reproductive model to investigate maternal age-related alterations in reproductive organs and embryos on Day 6 of pregnancy. Blood, ovaries, endometrium, and blastocysts from young (16-20 weeks) and advanced maternal age phase (>108 weeks, old) rabbits were analysed at the mRNA and protein levels to investigate the insulin-like growth factor (IGF) system, lipid metabolism, and stress defence system. Older rabbits had lower numbers of embryos at Day 6 of pregnancy. Plasma insulin and IGF levels were reduced, which was accompanied by paracrine regulation of IGFs and their receptors in ovaries and endometrium. Embryos adapted to hormonal changes as indicated by reduced embryonic IGF1 and 2 levels. Aged reproductive organs increased energy generation from the degradation of fatty acids, leading to higher oxidative stress. Stress markers, including catalase, superoxide dismutase 2, and receptor for advanced glycation end products were elevated in ovaries and endometrium from aged rabbits. Embryonic fatty acid uptake and ß-oxidation were increased in both embryonic compartments (embryoblast and trophoblast) in old rabbits, associated with minor changes in the oxidative and glycative stress defence systems. In summary, the insulin/IGF system, lipid metabolism, and stress defence were dysregulated in reproductive tissues of older rabbits, which is consistent with changes in embryonic metabolism and stress defence. These data highlight the crucial influence of maternal age on uterine adaptability and embryo development.

Ectopic Lipid Accumulation Correlates with Cellular Stress in Rabbit Blastocysts from Diabetic Mothers.

Maternal diabetes mellitus in early pregnancy leads to hyperlipidemia in reproductive tract organs and an altered embryonic environment. To investigate the consequences on embryonic metabolism, the effect of high environmental-lipid levels was studied in rabbit blastocysts cultured with a lipid mixture in vitro and in blastocysts from diabetic, hyperlipidemic rabbits in vivo. The gene and protein expression of marker molecules involved in lipid metabolism and stress response were analyzed. In diabetic rabbits, the expression of embryoblast genes encoding carnitine palmityl transferase 1 and peroxisome proliferator-activated receptors α and γ increased, whereas trophoblast genes encoding for proteins associated with fatty acid synthesis and ß-oxidation decreased. Markers for endoplasmic (activating transcription factor 4) and oxidative stress (nuclear factor erythroid 2-related factor 2) were increased in embryoblasts, while markers for cellular redox status (superoxide dismutase 2) and stress (heat shock protein 70) were increased in trophoblasts from diabetic rabbits. The observed regulation pattern in vivo was consistent with an adaptation response to the hyperlipidemic environment, suggesting that maternal lipids have an impact on the intracellular metabolism of the preimplantation embryo in diabetic pregnancy and that embryoblasts are particularly vulnerable to metabolic stress.

Quantitative proteomics and in-cell cross-linking reveal cellular reorganisation during early neuronal differentiation of SH-SY5Y cells.

The neuroblastoma cell line SH-SY5Y is commonly employed to study neuronal function and disease. This includes cells grown under standard conditions or differentiated to neuron-like cells by administration of chemical reagents such as retinoic acid (RA) or phorbol-12-myristate-13-acetate (PMA). Even though SH-SY5Y cells are widely explored, a complete description of the resulting proteomes and cellular reorganisation during differentiation is still missing. Here, we relatively quantify the proteomes of cells grown under standard conditions and obtained from two differentiation protocols employing RA or a combination of RA and PMA. Relative quantification and KEGG pathway analysis of the proteins reveals the presence of early differentiating cells and provides a list of marker proteins for undifferentiated and differentiated cells. For characterisation of neuronal sub-types, we analyse expression of marker genes and find that RA-differentiated cells are acetylcholinergic and cholinergic, while RA/PMA-differentiated cells show high expression of acetylcholinergic and dopaminergic marker genes. In-cell cross-linking further allows capturing protein interactions in different cellular organelles. Specifically, we observe structural reorganisation upon differentiation involving regulating protein factors of the actin cytoskeleton.

Embryonic fatty acid metabolism in diabetic pregnancy: the difference between embryoblasts and trophoblasts.

During the first days of development the preimplantation embryo is supplied with nutrients from the surrounding milieu. Maternal diabetes mellitus affects the uterine microenvironment, leading to a metabolic adaptation processes in the embryo. We analysed embryonic fatty acid (FA) profiles and expression of processing genes in rabbit blastocysts, separately in embryoblasts (EBs) and trophoblasts (TBs), to determine the potential consequences of maternal diabetes mellitus on intracellular FA metabolism. Insulin-dependent diabetes was induced by alloxan in female rabbits. On Day 6 post coitum, FA profiles in blastocysts (EB, TB and blastocoel fluid) and maternal blood were analysed by gas chromatography. The expression levels of molecules involved in FA elongation (fatty acid elongases, ELOVLs) and desaturation (fatty acid desaturases, FADSs) were measured in EB and TB. Maternal diabetes mellitus influenced the FA profile in maternal plasma and blastocysts. Independent from metabolic changes, rabbit blastocysts contained a higher level of saturated fatty acids (SFAs) and a lower level of polyunsaturated fatty acids (PUFAs) compared to the FA profile of the maternal plasma. Furthermore, the FA profile was altered in the EB and TB, differently. While SFAs (palmitic and stearic acid) were elevated in EB of diabetic rabbits, PUFAs, such as docosahexaenoic acid, were decreased. In contrast, in the TB, lower levels of SFAs and higher levels of oleic acid were observed. EB and TB specific alterations in gene expression were found for ELOVLs and FADSs, key enzymes for FA elongation and desaturation. In conclusion, maternal diabetes mellitus alters embryonic FA metabolism differently in EB and TB, indicating a lineage-specific metabolic adaptive response.

Metabolic Profiling in Blastocoel Fluid and Blood Plasma of Diabetic Rabbits.

Metabolic disorders of the mother adversely affect early embryo development, causing changes in maternal metabolism and consequent alterations in the embryo environment in the uterus. The goal of this study was to analyse the biochemical profiles of embryonic fluids and blood plasma of rabbits with and without insulin-dependent diabetes mellitus (DT1), to identify metabolic changes associated with maternal diabetes mellitus in early pregnancy. Insulin-dependent diabetes was induced by alloxan treatment in female rabbits 10 days before mating. On day 6 post-coitum, plasma and blastocoel fluid (BF) were analysed by ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) (Metabolon Inc. Durham, NC, USA). Metabolic datasets comprised a total of 284 and 597 compounds of known identity in BF and plasma, respectively. Diabetes mellitus had profound effects on maternal and embryonic metabolic profiles, with almost half of the metabolites changed. As predicted, we observed an increase in glucose and a decrease in 1,5-anhydroglucitol in diabetic plasma samples. In plasma, fructose, mannose, and sorbitol were elevated in the diabetic group, which may be a way of dealing with excess glucose. In BF, metabolites of the pentose metabolism were especially increased, indicating the need for ribose-based compounds relevant to DNA and RNA metabolism at this very early stage of embryo development. Other changes were more consistent between BF and plasma. Both displayed elevated acylcarnitines, body3-hydroxybutyrate, and multiple compounds within the branched chain amino acid metabolism pathway, suggesting that lipid beta-oxidation is occurring at elevated levels in the diabetic group. This study demonstrates that maternal and embryonic metabolism are closely related. Maternal diabetes mellitus profoundly alters the metabolic profile of the preimplantation embryo with changes in all subclasses of metabolites.

Glyoxalase 1 expression is downregulated in preimplantation blastocysts of diabetic rabbits.

In a diabetic pregnancy, an altered maternal metabolism led to increased formation of reactive α-dicarbonyls such as glyoxal (GO) and methylglyoxal (MGO) in the reproductive organs and embryos. The enzyme glyoxalase (GLO) 1 detoxifies reactive α-dicarbonyls thus protecting cells against malfunction or modifications of proteins by advanced glycated end products (AGEs). The aim of this study was to analyse the influence of a maternal insulin-dependent diabetes mellitus (IDD) on GLO1 expression and activity in preimplantation embryos in vivo and human trophoblast cells (Ac-1M88) in vitro. Maternal diabetes was induced in female rabbits by alloxan before conception and maintained during the preimplantation period. GLO1 expression and activity were investigated in 6-day-old blastocysts from healthy and diabetic rabbits. Furthermore, blastocysts and human trophoblast cells were exposed in vitro to hyperglycaemia, GO and MGO and analysed for GLO1 expression and activity. During gastrulation, GLO1 was expressed in all compartments of the rabbit blastocyst. Maternal diabetes decreased embryonic GLO1 protein amount by approx. 30 per cent whereas the enzymatic activity remained unchanged, indicating that the specific GLO1 activity increases along with metabolic changes. In in vitro cultured embryos, neither hyperglycaemia nor MGO and GO had an effect on GLO1 protein amount. In human trophoblast cells, a stimulating effect on the GLO1 expression was shown in the highest GO concentration, only. Our data show that maternal diabetes mellitus affects the specific activity of GLO1, indicating that GLO1 was post-translationally modified due to changes in metabolic processes in the preimplantation embryos.

Adipose-Derived Stem/Stromal Cells Recapitulate Aging Biomarkers and Show Reduced Stem Cell Plasticity Affecting Their Adipogenic Differentiation Capacity.

Stromal mesenchymal stem cells (MSCs) have the capability to self-renew and can differentiate into multiple cell types of the mesoderm germ layer, but their properties are affected by molecular aging mechanisms. MSCs can be obtained from adipose tissue termed as adipose-derived stem/stromal cells (ASCs) representing a promising tool for studying age-related diseases in detail. ASCs from young (16 weeks) and old (>108 weeks) rabbits were successfully isolated and propagated. ASCs showed the typical morphology and stained positive for CD105, Vimentin, Collagenase 1A, and negative for CD14, CD90, and CD73, demonstrating their mesenchymal origin. ASCs expressed MSC markers, including MYC, KLF4, CHD1, REST, and KAT6A, whereas pluripotency-related genes, such as NANOG, OCT4, and SOX2, were not expressed. Aged ASCs showed altered protein and mRNA levels of APOE, ATG7, FGF2, PTEN, and SIRT1. Adipogenic differentiation of old visceral ASCs was significantly decreased compared with young visceral ASCs. We successfully established rabbit ASC cultures representing an in vitro model for the analysis of stem cell aging mechanisms. ASCs, obtained from old female rabbits, showed age- and source-specific alteration due to aging of the donor. Stem cell plasticity was altered with age as shown by reduced adipogenic differentiation capacity.

Trophoblastic microRNAs are downregulated in a diabetic pregnancy through an inhibition of Drosha.

MicroRNAs are promising biological markers for prenatal diagnosis. They regulate placental development and are present in maternal plasma. Maternal metabolic diseases are major risk factors for placental deterioration. We analysed the influence of a maternal insulin-dependent diabetes mellitus on microRNA expression in maternal plasma and in blastocysts employing an in vivo rabbit diabetic pregnancy model and an in vitro embryo culture in hyperglycaemic and hypoinsulinaemic medium. Maternal diabetes led to a marked downregulation of Dicer protein in embryoblast cells and Drosha protein in trophoblast cells. MiR-27b, miR-141 and miR-191 were decreased in trophoblast cells and in maternal plasma of diabetic rabbits. In vitro studies indicate, that maternal hyperglycaemia and hypoinsulinaemia partially contribute to the downregulation of trophoblastic microRNAs. As the altered microRNA expression was detectable in maternal plasma, too, the plasma microRNA signature could serve as an early biological marker for the prediction of trophoblast function during a diabetic pregnancy.

Comparative Analysis of AGE and RAGE Levels in Human Somatic and Embryonic Stem Cells under H2O2-Induced Noncytotoxic Oxidative Stress Conditions.

The accumulation of advanced glycation end products (AGEs) occurs in ageing and in many degenerative diseases as a final outcome of persistent oxidative stress on cells and organs. Environmental alterations taking place during early embryonic development can also lead to oxidative damage, reactive oxygen species (ROS) production, and AGE accumulation. Whether similar mechanisms act on somatic and embryonic stem cells (ESC) exposed to oxidative stress is not known; and therefore, the modelling of oxidative stress in vitro on human ESC has been the focus of this study. We compared changes in N ε -carboxymethyl-lysine (CML) advanced glycation end products and RAGE levels in hESC versus differentiated somatic cells exposed to H2O2 within the noncytotoxic range. Our data revealed that hESC accumulates CML and RAGE under oxidative stress conditions in different ways than somatic cells, being the accumulation of CML statistically significant only in somatic cells and, conversely, the RAGE increase exclusively appreciated in hESC. Then, following cardiac and neural differentiation, we observed a progressive removal of AGEs and at the same time an elevated activity of the 20S proteasome. We conclude that human ESCs constitute a unique model to study the consequence of an oxidative environment in the pluripotent cells of the embryo during the human preimplantation period.

Adiponectin stimulates lipid metabolism via AMPK in rabbit blastocysts.

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 ß-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.

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.

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.

Role of advanced glycation end products in cellular signaling.

Improvements in health care and lifestyle have led to an elevated lifespan and increased focus on age-associated diseases, such as neurodegeneration, cardiovascular disease, frailty and arteriosclerosis. In all these chronic diseases protein, lipid or nucleic acid modifications are involved, including cross-linked and non-degradable aggregates, such as advanced glycation end products (AGEs). Formation of endogenous or uptake of dietary AGEs can lead to further protein modifications and activation of several inflammatory signaling pathways. This review will give an overview of the most prominent AGE-mediated signaling cascades, AGE receptor interactions, prevention of AGE formation and the impact of AGEs during pathophysiological processes.

Tributyltin affects adipogenic cell fate commitment in mesenchymal stem cells by a PPARγ independent mechanism.

The food contaminant tributyltin (TBT) is an endocrine disrupting compound (EDC) promoting adipogenic differentiation in vitro and in vivo. Although prenatal TBT exposure has been shown to induce obesity, the underlying mechanisms and the role of the transcription factor PPARγ are not clarified yet. At different stages of adipogenesis, multipotent murine mesenchymal stem cells (MSC), C3H10T1/2, were exposed to TBT and analyzed for adipogenic differentiation, PPARγ promoter activation and PPARγ1, PPARγ2, Pref-1 and SOX9 expression. Depending on the exposure window, TBT promoted subsequent adipogenesis independently and dependently from PPARγ. In undifferentiated MSC, TBT exposure induced a transcriptional PPARγ-independent repression of Pref-1 and SOX9, which are both suppressors of adipogenic cell fate commitment. During hormonal induction TBT additionally enhanced adipogenic differentiation by PPARγ signaling. The impact of TBT on early cell fate development documents a novel mechanistic insight in the development of adipocytes derived from MSC and its susceptibility to EDC.

Adipogenic effects of a combination of the endocrine-disrupting compounds bisphenol A, diethylhexylphthalate, and tributyltin.

OBJECTIVE: The food contaminants bisphenol A (BPA), diethylhexylphthalate (DEHP), and tributyltin (TBT) are potent endocrine-disrupting compounds (EDC) known to interfere with adipogenesis. EDC usually act in mixtures and not as single compounds. The aim of this study was to investigate the effects of a simultaneous exposure of BPA, DEHP, and TBT on mesenchymal stem cell differentiation into adipocytes. METHODS: Multipotent murine mesenchymal stem cells (C3H10T1/2) were exposed to EDC mixtures in high concentrations, i.e. MIX-high (10 µmol/l BPA, 100 µmol/l DEHP, 100 nmol/l TBT), and in environmentally relevant concentrations, i.e. MIX-low (10 nmol/l BPA, 100 nmol/l DEHP, 1 nmol/l TBT). The exposure was performed either for the entire culture time (0-12 days) or at distinct stages of adipogenic differentiation. At day 12 of cell culture, the amount of adipocytes, triglyceride content (TG), and adipogenic marker gene expression were analyzed. RESULTS: MIX-high increased the development of adipocytes and the expression of adipogenic marker genes independently of the exposure window. The total TG amount was not increased. The low-concentrated EDC mixture had no obvious impact on adipogenesis. CONCLUSION: In EDC mixtures, the adipogenic effect of TBT and DEHP predominates single effects of BPA. Mixture effects of EDC are not deducible from single compound experiments.

Expression profile of fatty acid metabolism genes in preimplantation blastocysts of obese and non-obese mice.

BACKGROUND: Recent studies have disclosed a close relationship between maternal obesity, fetal metabolism and pre- and postnatal development. The lipid metabolism in preimplantation embryos is a possible target of metabolic programming. METHODS: 31 genes of beta-oxidation and fatty acid and cholesterol uptake, synthesis and regulation were analyzed in day 3.5 blastocysts from NZO (obese) and C57Bl/6 (normal weight) mice by RT-PCR and semiquantitative PCR. RESULTS: The most obvious difference between both strains was the lack of the RXR gamma transcript in NZO blastocysts. In adult NZO mice, RXR gamma is detectable in most tissues. In a semiquantitative analysis, a higher transcription rate of fatty acid transport protein 4 (p = 0.004) and a reduced transcript number of fatty acid synthase (p = 0.049) was found in NZO blastocysts. Cholesterol synthesis regulation was modified in NZO blastocysts, as indicated by the ratio of sterol regulatory element-binding protein (SREBP) 2 / insulin-induced gene 1 (Insig 1) (p = 0.001). CONCLUSION: In mouse blastocysts enzymes and signal molecules of fatty acid and cholesterol metabolism resemble those expressed postnatally. Distinct differences in transcription rates of genes between blastocysts from obese and non-obese mothers indicate that preimplantation embryo development is an early target for metabolic programming.

Rabbit as a reproductive model for human health.

The renaissance of the laboratory rabbit as a reproductive model for human health is closely related to the growing evidence of periconceptional metabolic programming and its determining effects on offspring and adult health. Advantages of rabbit reproduction are the exact timing of fertilization and pregnancy stages, high cell numbers and yield in blastocysts, relatively late implantation at a time when gastrulation is already proceeding, detailed morphologic and molecular knowledge on gastrulation stages, and a hemochorial placenta structured similarly to the human placenta. To understand, for example, the mechanisms of periconceptional programming and its effects on metabolic health in adulthood, these advantages help to elucidate even subtle changes in metabolism and development during the pre- and peri-implantation period and during gastrulation in individual embryos. Gastrulation represents a central turning point in ontogenesis in which a limited number of cells program the development of the three germ layers and, hence, the embryo proper. Newly developed transgenic and molecular tools offer promising chances for further scientific progress to be attained with this reproductive model species.

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.