Influence of maternal metabolism and parental genetics on fetal maldevelopment in diabetic rat pregnancy.

The purpose of this study was to investigate the influence of parental transgenerational genetics and maternal metabolic state on fetal maldevelopment in diabetic rat pregnancy. Rats from an inbred malformation-resistant (W) strain, and an inbred malformation-prone (L) strain, were cross-mated to produce two different F(1) hybrids, WL and LW. Normal (N) and manifestly diabetic (MD) WL and LW females were mated with normal males of the same F(1) generation to obtain WLWL and LWLW F(2) hybrids. Maternal diabetes increased malformation and resorption rates in both F(2) generations. MD-WLWL offspring had higher resorption rate but similar malformation rate compared with the MD-LWLW offspring. Malformed MD-WLWL offspring presented with 100% agnathia/micrognathia, whereas malformed MD-LWL offspring had 60% agnathia/micrognathia and 40% cleft lip and palate. The MD-WL dams showed increased ß-hydroxybutyrate levels and alterations in concentrations of several amino acids (taurine, asparagine, citrulline, cystine, glutamic acid, leucine, tyrosine, and tryptophan) compared with MD-LW dams. Fetal glyceraldehyde-3-phosphate dehydrogenase (Gapdh) activity and gene expression were more altered in MD-WLWL than MD-LWLW. Fetal gene expression of reactive oxygen species (ROS) scavenger enzymes was diminished in MD-WLWL compared with MD-LWLW. Glial cell line-derived neurotrophic factor and Ret proto-oncogene gene expression was decreased in both MD-WLWL and MD-LWLW fetuses, whereas increased bone morphogenetic protein 4 and decreased Sonic hedgehog homolog expression was found only in MD-LWLW fetuses. Despite identical autosomal genotypes, the WL and LW dams gave birth to offspring with markedly different malformation patterns. Together with fetal differences in enzymatic activity and expression of Gapdh, ROS scavengers, and developmental genes, these results may suggest a teratological mechanism in diabetic pregnancy influenced by maternal metabolism and parental strain epigenetics.

Genetic and environmental influence on diabetic rat embryopathy.

We assessed genetic and environmental influence on fetal outcome in diabetic rat pregnancy. Crossing normal (N) and manifestly diabetic (MD) Wistar Furth (W) and Sprague-Dawley (L) females with W or L males yielded four different fetal genotypes (WW, LL, WL, and LW) in N or MD rat pregnancies for studies. We also evaluated fetal outcome in litters with enhanced or diminished severity of maternal MD state, denoted MD(+)WL and MD(-)LW. The MDWW litters had less malformations and resorptions (0 and 19%) than the MDLL litters (17 and 30%). The MDWL litters (0 and 8%) were less maldeveloped than the MDLW litters (9 and 22%), whereas the MD(+)WL (3 and 23%) and MD(-)LW (1 and 17%) litters showed increased and decreased dysmorphogenesis (compared with MDWL and MDLW litters). The pregnant MDW rats had lower serum levels of glucose, fructosamine, and branched-chain amino acids than the pregnant MDL rats, whereas the pregnant MD(+)W and MD(-)L rats had levels comparable with those of the MDL and MDW rats, respectively. The 8-iso-PGF2α levels of the malformed MDLW offspring were increased compared with the nonmalformed MDLW offspring. Diabetes decreased fetal heart Ret and increased Bmp-4 gene expression in the MDLW offspring and caused decreased GDNF and Shh expression in the malformed fetal mandible of the MDLW offspring. We conclude that the fetal genome controls the embryonic dysmorphogenesis in diabetic pregnancy by instigating a threshold level for the teratological insult and that the maternal genome controls the teratogenic insult by (dys)regulating the maternal metabolism.

Diabetes in pregnancy. Skeletal malformations in the offspring of diabetic rats after intermittent withdrawal of insulin in early gestation.

Precise timing of the teratogenic period in diabetic pregnancy is of clinical importance since correction of the glucose intolerance during this period may protect the offspring from malformations. An experimental approach to elucidate this problem with regard to skeletal development was made in groups of pregnant streptozotocin-diabetic rats (MDI), which were treated with daily insulin injections except for a 2-day period in the first half of pregnancy. The degree of metabolic derangement was estimated by measurements of serum glucose concentrations. During the insulin-free period, the rats showed severe hyperglycemia (greater than 20 mM) while during ongoing insulin treatment, only brief periods of hyper- or hypoglycemia were observed. Insulin treatment was withdrawn successively between gestational days 3 and 12. Control groups consisted of normal pregnant rats (N) or pregnant rats with manifest diabetes (MD) without insulin treatment. The serum glucose levels of the N animals were below 6 mM while those of the MD animals were above 25 mM throughout pregnancy. Skeletal malformations in the viable offspring were recorded on gestational day 20 after Alizarin staining of calcified ossification centers, which also allowed an estimate of skeletal development as a whole. Untreated diabetes in the MD rats induced a high rate of fetal resorptions, a decrease in fetal weight and viability, as well as retardation of skeletal development. Intermittent insulin treatment in the MDI rats ameliorated, but did not abolish, these changes. In the MD group 9 of 48 viable fetuses showed severe malformations of either the lower jaw (micrognathia) or of the lumbosacral region (caudal dysgenesis).(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of cellular retinoic acid-binding protein I during odontogenesis in the rat incisor.

Retinoids are important molecules in various aspects of embryological development. Here the distribution of cellular retinoic acid-binding protein I (CRABPI) was studied in the continuously growing incisor of adult rats using an affinity-purified rabbit polyclonal antibody. CRABPI was present throughout the presecretory and secretory ameloblast layer. The protein disappeared from that layer during its maturation phase. The adjacent dental mesenchyme of the developing pulp stained positively for CRABPI, especially in the layer immediately beneath the fully differentiated odontoblasts. Little CRABPI was present in the odontoblast layer itself. The distribution of CRABPI, both in the undifferentiated basal region of the incisor tooth and associated with the cells during hard-tissue formation, suggests a role for this molecule during differentiation and hard-tissue genesis.

Metabolism in vitro of cartilage proteoglycans in rat (pre)chondrocytes from different embryonic regions.

Diabetes in the mother may cause disturbances in the chondrocyte development in the embryo. A rat model was used to investigate whether this was reflected in the production of proteoglycans by cells from two embryonic regions. One of these regions is resistant (limb bud) and the other susceptible (mandibular arch) to malformation in diabetic pregnancy. Chondroitin sulphate proteoglycans from cultures of day-12 rat embryo limb bud and mandibular arch chondrocytes were extracted with guanidine-HCl and analyzed by gel chromatography after in vitro 35S-sulphate-labeling. Two sizes of proteoglycans (Kav 0.26 and 0.66 on CL-2B Sepharose) were found in both types of chondrocytes and in all media. The polysaccharide chain length was the same (Kav 0.36 on CL-6B Sepharose) for both proteoglycans. Elevated levels of D-glucose or beta-hydroxybutyric acid had no effect on either proteoglycan size or proportion, nor on polysaccharide chain length. However, there were differences (in all culture conditions) between limb bud and mandibular arch cultures in that the larger proteoglycan accounted for 80% of total radioactivity in the limb bud cultures, 53% in the mandibular arch cultures, and only 25-29% in the media from both types of cultures. Furthermore, different ratios between radioactive proteoglycans in medium and matrix suggested markedly different efficiencies for matrix formation in the two cell types. These findings indicate differences in the metabolism of the proteoglycans in these two cell types which may be related to the induction of mandibular malformation in diabetic pregnancy.

In vitro effects of glucose and growth factors on limb bud and mandibular arch chondrocytes maintained at various serum concentrations.

In human and experimental diabetic pregnancy there is an increased risk of congenital malformation in the offspring. Some malformations involve growth retardation and altered chondrocyte differentiation, suggesting that a diabetic milieu may modify embryonic cell replication and the development of (pre)chondrocytes. The aim of the present work was to study the effects of a diabetes-like environment in vitro on the growth and differentiation of rat chondrocytes in the presence of specific growth factors and different concentrations of serum. This was performed with a modified micromass culture system of embryonic (pre)chondrocytes from the limb bud and mandibular arch areas using medium supplemented with different glucose concentrations and with serum from diabetic rats. An elevated ambient glucose concentration inhibited the growth of mature chondrocytes in vitro, and this effect was diminished in a serum-rich culture milieu. The (pre)chondrocytes exhibited a marked dependence on the serum level in the culture medium for optimal in vitro development. Diabetic rat serum had the lowest stimulatory capacity of the three different types tested (at similar glucose concentrations), suggesting a deficiency of growth-stimulating factor(s) rather than the presence of inhibiting factor(s) in this type of serum. One of the deficient factor(s) in diabetic rat serum may be similar to IGF-II, but a combined deficiency of several growth-stimulating agents is likely to be present. Chondrocytes originating from the mandibular arch in general appeared more sensitive to MSA and IGF-II than those from the limb buds. The present observations support the notion that while diabetes-induced hyperglycemia in the conceptus contributes to severe growth retardation of the mandibular arch, additional factors also play a role.

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.

Effects of D-glucose and beta-hydroxybutyric acid on the in vitro development of (pre)chondrocytes from embryos of normal and diabetic rats.

In order to elucidate cellular mechanisms causing skeletal malformations in offspring of diabetic rats we studied the incorporation of thymidine and sulphate into embryonic (pre)chondrocytes exposed to increased levels of D-glucose and beta-hydroxybutyric acid for six days in vitro. The (pre)chondrocytes were prepared from embryos of normal or diabetic rats of a malformation-prone strain or from embryos of normal rats of a non-malformation-prone strain. Diabetic female rats of the former strain are known to produce a high proportion of offspring with mandibular and lumbosacral malformations. Increased beta-hydroxybutyric acid caused decreased thymidine incorporation in all types of chondrocytes, and decreased sulphate incorporation in limb bud cells from embryos of normal rats from both strains. Elevated D-glucose levels yielded a slight decrease in thymidine incorporation in mandibular arch cells from embryos of normal rats of the malformation-prone strain, and a marked decrease of both sulphate and thymidine incorporation in mandibular arch cells from embryos of diabetic rats of this strain. The observations suggest that elevated levels of D-glucose or beta-hydroxybutyric acid are able to inhibit the differentiation and growth of (pre)-chondrocytes and illustrate a selective sensitivity of mandibular arch (pre)chondrocytes to a diabetic environment. The data are compatible with the view that both D-glucose and beta-hydroxybutyric acid may cause aberrations in the development of rat mandibular arch chondrocytes, suggesting a role for these compounds in diabetic teratogenesis.

Biosynthesis of 9-cis-retinoic acid in vivo. The roles of different retinol dehydrogenases and a structure-activity analysis of microsomal retinol dehydrogenases.

Retinoic acid is generated by a two-step mechanism. First, retinol is converted into retinal by a retinol dehydrogenase, and, subsequently, retinoic acid is formed by a retinal dehydrogenase. In vitro, several enzymes are suggested to act in this metabolic pathway. However, little is known regarding their capacity to contribute to retinoic acid biosynthesis in vivo. We have developed a versatile cell reporter system to analyze the role of several of these enzymes in 9-cis-retinoic acid biosynthesis in vivo. Using a Gal4-retinoid X receptor fusion protein-based luciferase reporter assay, the formation of 9-cis-retinoic acid from 9-cis-retinol was measured in cells transfected with expression plasmids encoding different combinations of retinol and retinal dehydrogenases. The results suggested that efficient formation of 9-cis-retinoic acid required co-expression of retinol and retinal dehydrogenases. Interestingly, the cytosolic alcohol dehydrogenase 4 failed to efficiently catalyze 9-cis-retinol oxidation. A structure-activity analysis showed that mutants of two retinol dehydrogenases, devoid of the carboxyl-terminal cytoplasmic tails, displayed greatly reduced enzymatic activities in vivo, but were active in vitro. The cytoplasmic tails mediate efficient endoplasmic reticulum localization of the enzymes, suggesting that the unique milieu in the endoplasmic reticulum compartment is necessary for in vivo activity of microsomal retinol dehydrogenases.

Malformations in offspring of diabetic rats: morphometric analysis of neural crest-derived organs and effects of maternal vitamin E treatment.

BACKGROUND: We have previously reported on a malformation-prone Sprague-Dawley rat substrain (U), which presents a high frequency of micrognathia in the offspring of diabetic mothers. This malformation is related to impaired development of the cranial neural crest cells (NCC); the defect may be prevented by antioxidative treatment of the mother. METHODS: We have therefore investigated whether fetuses of diabetic rats display other malformations associated with altered cranial NCC development and whether maternal vitamin E supplementation may affect such malformations. RESULTS: Fetuses of diabetic rats showed low-set external ears, severely malformed Meckel's cartilage, small thyroid and thymus, and absence of parathyroid glands. Cardiac anomalies were frequently observed, including rightward displacement of the aorta, double outlet right ventricle (DORV), persistent truncus arteriosus (PTA) combined with ventricular septal defects due to a malaligned outlet septum. The malformations in the outflow tract included abnormalities of the great arteries; right-sided aortic arch/descending aorta, and double aortic arches. These defects tended to occur together within individual fetuses. Maternal dietary treatment with 2% vitamin E markedly reduced the severity of the malformations. CONCLUSIONS: The phenotypic appearance of these defects is strikingly similar to the DiGeorge anomaly in humans, which has been found in children of diabetic mothers together with an overrepresentation of PTA and DORV. The malformations associated with defective NCC development in the offspring of diabetic U rats show several morphological similarities to those in humans; hence the teratogenic mechanisms may be similar and accessible for study.