Altered gene expression in rat cranial neural crest cells exposed to a teratogenic glucose concentration in vitro: paradoxical downregulation of antioxidative defense genes.

BACKGROUND: Diabetic pregnancy is associated with increased risk of malformation in the infant. Diabetes-induced anomalies of the face and heart are strongly correlated with neural crest cell (NCC) maldevelopment. We aimed to study glucose-induced alterations of mRNA levels in cranial and trunk NCCs isolated from rat embryos with increased risk of developing mandibular and cardiac malformations in diabetic pregnancy. METHODS: Inbred Sprague-Dawley rat embryos were used for NCC isolation from neural tube explants. The migrating cells were exposed to 5.5 or 30 mmol/l glucose concentration for 48 hr, harvested, and prepared for gene expression measurement by RT-PCR or immunostaining with either distal-less (Dlx) or AP-2-α antibodies. RESULTS: Evaluation of the immunostained slides showed that approximately 75% of the cells were of NCC origin. Exposure to 30 mM glucose decreased mRNA levels of Copper-Zinc superoxide dismutase, manganese superoxide dismutase, extracellular superoxide dismutase, Catalase, Gpx-1, Nrf2, poly-ADP ribose polymerase, B-cell leukemia/lymphoma protein 2, and ß-Catenin genes in cranial neural crest explant cultures. In addition, Pax-3, Pax-6, Wnt3a, and Apc mRNA levels were decreased by high glucose exposure in both cranial and trunk neural crest explant cultures. CONCLUSION: Cranial NCCs diminish their mRNA levels of antioxidative enzymes and the Nrf2 response factor, as well as the antiapoptotic B-cell leukemia/lymphoma protein 2 gene, in response to increased ambient glucose concentration. Furthermore, both cranial and trunk NCC decrease the mRNA levels of the transcription factors Pax-3 and Pax-6, as well as key components of the Wnt pathway. These patterns of glucose-altered gene expression in a developmentally important cell population may be of etiological importance for NCC-associated malformations in diabetic pregnancy.

Fluctuating asymmetry in fetuses of diabetic rhesus macaques.

This study examines dental fluctuating asymmetry (FA) in two samples of fetal rhesus monkeys, one composed of 19 fetuses from diabetic mothers (FDM) and the other of 20 fetuses from nondiabetic mothers. Seventeen measurements were taken on the deciduous dentition of right and left mandibles. The degree of FA was assessed by comparing FDM to fetuses of normal mothers by correlation between right and left sides, and analysis of variation differences between right and left sides. Significant FA was found for three traits based on the correlation between right and left sides and for seven traits by the between-treatment ratio of variance between sides. Distal teeth, both within and outside of a morphologic field, exhibit significantly greater FA than mesial teeth. Our results support the hypothesis that developmental instability is detectable by dental FA.

Effects of maternal dietary protein on the mandibular growth of fetuses from diabetic dams.

The effects of maternal diabetes on the mandibles in the fetuses of dams given a normal (20% protein) diet vs those given a high protein (40% protein) diet were studied. On day 9, the controls, groups 1 and 3, were injected with citrate buffer and fed a 20% and 40% protein diet, respectively. Groups 2 and 4 were injected with 40 mg/kg B.W. of streptozotocin and pair-fed with groups 1 and 3 with a 20% and 40% protein diet, respectively. On day 22, fetuses were removed. Dissected mandibles were weighed and analyzed for DNA, protein, Ca and hydroxyproline contents. Some dams in each group were injected with either 45Ca or 14C proline to study 45Ca uptake and collagen synthesis. The body and mandibular weights of the fetuses from diabetic (D) dams were smaller than those of the non-diabetic (ND) group in both diets. The protein and hydroxyproline contents of the mandible in the D 20% group was less than the ND. Hydroxyproline content from the D 40% group was less than the ND. The Ca content in the D 40% group was less than the ND. The present study indicates that metabolism in the bone of fetuses whose D dams were fed a high protein diet is affected beneficially in some parameters but not in others.

Regionally disturbed production of cartilage proteoglycans in malformed fetuses from diabetic rats.

Fetuses from normal and manifestly diabetic rats were obtained on pregnancy day 20. The fetuses from the diabetic rats were of normal or malformed morphology. Three tissue groups were dissected free; costal cartilage, the hard tissue of the rear, and of the frontal portion of the mandible. These tissues were maintained in vitro for 24 h during which time they were labelled with [35S]sulphate. After the culture period the tissues were extracted with guanidine HCl and the resulting residues were further extracted with alkali. The culture medium was saved and its macromolecular content was compared to that of the extracts. The proteoglycans recovered in all extracts eluted at two distinct positions after chromatography on a Sepharose CL-2B column (peak I: Kav approximately 0.4, and peak II: Kav approximately 0.8), but the elution patterns were markedly different in extracts from various tissues. Thus, in rib cartilage, the majority of the labelled proteoglycans were located in peak I (approximately 90%) with no difference between extracts of fetuses from normal and diabetic pregnancies. In extracts of mandibular cartilaginous tissue from normal rat offspring the peak I percentage (60-80%) was lower than in the rib cartilage extracts. In the extracts from the frontal portion of malformed mandibles of fetuses of diabetic rats, the peak I percentage (35 +/- 21%) was the lowest of all recorded and the only one to significantly differ from the other percentages in its (the frontal mandible) group. The results show an association between a congenital malformation, micrognathia, and a disturbance in the production of chondroitin sulphate proteoglycans in the malformed region.

Encapsulated beta-islet cells as a bioartificial pancreas to treat insulin-dependent diabetes during pregnancy.

OBJECTIVE: Our purpose was to determine the effectiveness of the bioartificial pancreas technique in correcting (1) maternal carbohydrate metabolism and (2) fetal malformation rates in a pregnant diabetic animal model. STUDY DESIGN: Insulin secretion from encapsulated rat islets cultured in the presence of homologous rat prolactin was determined and compared with that of controls. Streptozotocin-induced diabetic Balb/c mice were then transplanted with rat islet cells encapsulated within alginate microbeads and were then bred. Blood glucose determinations were made after transplantation and throughout gestation. Pups were delivered by cesarean section on day 19 of gestation. Outcome parameters from the transplanted study animals were compared with those of nondiabetic controls and untreated diabetic animals. RESULTS: Insulin secretion was increased twofold in encapsulated rat islets exposed to prolactin compared with control values. Throughout gestation maternal weights and blood, glucose levels of transplanted animals were similar to those of nondiabetic controls. A fetal malformation rate of only 1.4% was observed in the pups from transplanted animals. CONCLUSIONS: Transplanted encapsulated islets are capable of normalizing maternal carbohydrate metabolism in a pregnant diabetic animal model. This therapy, if instituted before conception, also appears to eliminate the increase in fetal malformations seen in diabetic pregnancies.