Hyperglycaemia in vitro alters the proliferation and mitochondrial activity of the choriocarcinoma cell lines BeWo, JAR and JEG-3 as models for human first-trimester trophoblast

Abstract

Despite considerable advances in the treatment of diabetes in the past decades, diabetic pregnancies can still be associated with fetal growth disturbances even when an optimized and intensive metabolic control has been achieved. Macrosomia of the fetus is a common and well-known consequence of maternal diabetes with an increased obstetric risk. The increase in fetal fat mass that accounts for the fetal overweight [1] is the result of fetal hyperinsulinism as a consequence of maternal and, therefore, fetal hyperglycaemia [2]. According to the dynamics of fetal growth, macrosomia is caused by metabolic alterations or hyperglycaemic insults predominantly in later stages of pregnancy [3,4]. In contrast diabetes-associated metabolic derangements in the first trimester of pregnancy can lead to malformations [5] and to delayed growth of the developing embryo [6,7]. Whereas the molecular mechanisms underlying embryo malformations have received enormous attention, no data are available to explain early fetal growth delay. Because of the close link between placental and fetal growth, early intrauterine growth delay could be caused by a reduced growth of the pla- Diabetologia (2001) Abstract Aims/hypothesis. Early intrauterine growth delay in diabetes could be caused by a reduced growth of the placenta. Our study investigates whether hyperglycaemia in vitro reduces trophoblast proliferation. Methods. First-trimester trophoblast cell models (BeWo, JAR and JEG-3 choriocarcinoma cells) were cultured for 24 and 48 h with 5.5 mmol/l d-glucose, 25 mmol/l d-glucose (hyperglycaemia) and with an osmotic control. Cell number, total protein and nucleic acid content and mitochondrial activity (tetrazolium salt assay) were measured, the cell cycle analysed (FACS, cyclin B1 levels) and apoptosis (Annexin-V) measured.Results. In BeWo cells hyperglycaemia reduced cell number, protein, nucleic acid and cyclin B1 levels. The reduced G 2 /M and increased G 0 /G 1 population after 24 h reflects growth arrest at G 0 /G 1 . In JAR cells after 24 h the population was arrested in G 0 /G 1 , whereas after 48 h the G 0 /G 1 block was abrogated and the cells were arrested at G 2 /M. The net effect was an unchanged cell number. In JEG-3 cells hyperglycaemia resulted in fewer cells after 24 h but not after 48 h indicating some adaptation. Mitochondrial activity was either stimulated (BeWo) or reduced (JAR, JEG-3) under hyperglycaemia. Some of these effects were also induced by hyperosmolarity alone. Conclusion/interpretation. Hyperglycaemia has the potential to inhibit the proliferation of first-trimester trophoblast cell models. The mechanisms leading to growth arrest and to changes in mitochondrial activity are complex and depend on differentiation. We hypothesise a hyperglycaemia-induced impairment of placental growth in the first trimester of a poorly controlled diabetic pregnancy. [Diabetologia (2001) 44: 209±219]

HbA1c and birthweight in women with pre-conception type 1 and type 2 diabetes: a population-based cohort study

“…We speculate that high glucose levels in early pregnancy may harm placental development and thus the capacity for fetal growth, such that the effect of hyperglycaemia in later pregnancy is permanently attenuated. The presence of microvascular disease, in particular in combination with first-trimester hyperglycaemia, can inhibit trophoblast proliferation, thereby reducing placental growth and impairing uteroplacental function, which may result in subsequent intrauterine growth restriction [10,37]. In our study, microvascular complications (pre-pregnancy retinopathy and/or nephropathy) in women with diabetes were associated with lower birthweight.…”

Differential glucocorticoid effects on proliferation and invasion of human trophoblast cell lines

“…This method has been recently used to demonstrate the effects of hyperglycaemia on choriocarcinoma cells (Weiss et al 2001).…”

“…Proteins (40 mg) of adherent cells were subjected to SDS-PAGE and immunoblotting as described (Weiss et al 2001). The blotting membranes were incubated (overnight, 4 8C) with either a polyclonal anti-GC receptor antibody (1:500; P-20 Santa Cruz; Santa Cruz, CA) or a monoclonal anti-cyclin B1 antibody (1:500; Pharmingen, San Diego, CA).…”

Glucose Regulates Insulin Mitogenic Effect by Modulating SHP-2 Activation and Localization in JAr Cells

“…These cells maintain in culture many features characteristic of human first trimester trophoblast, including chorionic gonadotropin and progesterone secretion (21). Because of these features, JAr cells have been widely used to investigate glucose and insulin action in placenta (19,20). We found that insulin does not induce proliferation of JAr cells when they are maintained in low glucose medium.…”