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Pregestational diabetes is highly associated with increased risk of birth defects. We previously reported that the expression of Cyp26a1, the major catabolizing enzyme for controlling retinoic acid (RA) homeostasis, is significantly down-regulated in embryos of diabetic mice, thereby increasing the embryo's susceptibility to malformations caused by RA dysregulation. However, the underlying mechanism for the down-regulation of Cyp26a1 remains unclear. This study aimed to investigate whether elevated maternal blood glucose in the diabetic milieu is a critical factor for the altered Cyp26a1 expression. Streptozotozin-induced diabetic pregnant mice were treated with phlorizin (PHZ) to reduce blood glucose concentrations via induction of renal glucosuria. Embryonic Cyp26a1 expression level, RA catabolic activity and susceptibility to various RA-induced abnormalities were examined. To test the dose-dependent effect of glucose on Cyp26a1 level, early head-fold stage rat embryos of normal pregnancy were cultured in vitro with varying concentrations of D-glucose, followed by quantification of Cyp26a1 transcripts. We found that Cyp26a1 expression, which was down-regulated in diabetic pregnancy, could be normalized under reduced maternal blood glucose level, concomitant with an increase in RA catabolic activity in embryonic tissues. Such normalization could successfully reduce the susceptibility to different RA-induced malformations including caudal regression, cleft palate and renal malformations. The expression level of Cyp26a1 in the embryo was inversely correlated with D-glucose concentrations. Diabetic patients suffer from retinopathy, dermopathy, male infertility and increased cancer risk. Coincidentally, RA dysregulation is also associated with these health problems. Our results provided evidence that elevated glucose can down-regulate Cyp26a1 expression level and disturb RA homeostasis, shedding light on the possibility of affecting the health of diabetic patients via a similar mechanism., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The effect of all-trans retinoic acid (RA) on embryogenesis is tissue specific and highly concentration dependent. Using a liquid chromatography/mass spectrometry-based method to quantify trace amounts of RA in embryonic tissue requires expensive specialist facilities. Here, we describe the use of a RA response element (RARE)-lacZ reporter cell-based method, which is simple and cost effective, to measure RA levels in small pieces of tissue from the embryo. We further apply this method to quantitatively assay activities of RA-synthesizing and RA-catabolizing enzymes, the key regulators of RA bioavailability in tissues and developing organs of the embryo.

Aims/hypothesis: We recently showed in mice that maternal diabetes increases embryonic susceptibility to caudal regression induced by vitamin A metabolite retinoic acid. Here we tested whether in the maternal diabetic milieu hyperglycaemia is the critical factor responsible for mediating this increased susceptibility., Methods: Non-diabetic pregnant mice were made hyperglycaemic by subcutaneous injections of glucose at regular intervals. Conversely, diabetic pregnant mice were treated with phlorizin to induce renal glucosuria and thus reduce blood glucose concentrations. Pregnant mice were treated with retinoic acid and the extent of caudal regression in mouse embryos, measured in terms of the ratio of tail length to crown-rump length was assessed. Embryos were also examined for Wnt-3a expression and cell death., Results: Embryos of mice treated with glucose had a greater extent of caudal regression induced by retinoic acid than saline-treated controls, with enhanced down-regulation of Wnt-3a expression and exacerbated cell death specifically at the caudal end of the embryo. Embryos of diabetic mice treated with phlorizin had a similar extent of caudal regression to embryos of non-diabetic mice after treatment with retinoic acid., Conclusions/interpretation: Hyperglycaemia increases embryonic susceptibility to caudal regression induced by retinoic acid, with the underlying cellular and molecular changes closely mimicking those that occur in maternal diabetes. Reduction of blood glucose concentrations in diabetic mice completely abolishes this increased susceptibility to retinoic acid. These results suggest that in maternal diabetes hyperglycaemia is the critical factor responsible for potentiating the teratogenic effect of retinoic acid.