Nutrient Transporter Gene Expression in the Early Conceptus—Implications From Two Mouse Models of Diabetic Pregnancy

Abstract

Maternal diabetes in early pregnancy increases the risk for birth defects in the offspring, particularly heart, and neural tube defects. While elevated glucose levels are characteristic for diabetic pregnancies, these are also accompanied by hyperlipidemia, indicating altered nutrient availability. We therefore investigated whether changes in the expression of nutrient transporters at the conception site or in the early post-implantation embryo could account for increased birth defect incidence at later developmental stages. Focusing on glucose and fatty acid transporters, we measured their expression by RT-PCR in the spontaneously diabetic non-obese mouse strain NOD, and in pregnant FVB/N mouse strain dams with Streptozotocin-induced diabetes. Sites of expression in the deciduum, extra-embryonic, and embryonic tissues were determined by RNAscope in situ hybridization. While maternal diabetes had no apparent effects on levels or cellular profiles of expression, we detected striking cell-type specificity of particular nutrient transporters. For examples, Slc2a2/Glut2 expression was restricted to the endodermal cells of the visceral yolk sac, while Slc2a1/Glut1 expression was limited to the mesodermal compartment; Slc27a4/Fatp4 and Slc27a3/Fatp3 also exhibited reciprocally exclusive expression in the endodermal and mesodermal compartments of the yolk sac, respectively. These findings not only highlight the significance of nutrient transporters in the intrauterine environment, but also raise important implications for the etiology of birth defects in diabetic pregnancies, and for strategies aimed at reducing birth defects risk by nutrient supplementation.

Aberrant lipid accumulation in the mouse visceral yolk sac resulting from maternal diabetes and obesity

“…Yet, their expression did not significantly differ between metabolic conditions. When interrogated for all genes with Slc (solute carrier) nomenclature, this dataset also did not reveal significant differences for over 200 nutrient transporter genes expressed at this stage (Kappen et al, 2022). Taking together our quantitative and qualitative data, they do not provide evidence that altered gene expression in lipid-related pathways could be invoked as causes for the excess lipid accumulation in any of our three experimental models.…”

“…Thus, lipids are either taken up from the maternal circulation, where they are in excess supply due to maternal hyperlipidemia in diabetic pregnancies, or they would have to be synthesized in visceral yolk sac from the excess msternal glucose under hyperglycemic conditions. We have previously shown that in addition to Glut3, the visceral yolk sac expresses the highcapacity glucose transporter Glut2, specifically in the endodermal cell layer (Kappen et al, 2022), lending plausibility to excessive glucose uptake. We here show that lipid transporters ApoA1, ApoB, SR-B1 and Cubilin are also expressed in the visceral yolk sac, with Cubilin and SR-B1 restricted to the endodermal layer, while ApoA1 and ApoB are also found in the mesodermal compartment of the visceral yolk sac, consistent with prior literature (Shi and Heath, 1984;Farese et al, 1996;Terasawa et al, 1999;Strope et al, 2004;Santander et al, 2013).…”

“…In order to model the hyperlipidemia that is characteristically associated with diabetic pregnancy, we also investigated pregnancies in FVB/N mice fed a high-fat-high-sucrose diet for 4 weeks prior to mating (Salbaum and Kappen, 2012). The specific focus on yolk sac was prompted by our earlier findings of layer-specific expression of glucose and fatty acid transporters in visceral yolk sac endodermal and mesodermal cells (Kappen et al, 2022). Since expression of these transporters at the mRNA level was unaffected by exposure to maternal diabetes in decidua, embryos, and yolk sacs, we hypothesized that abnormalities in nutrient uptake, processing and/or transport caused by the exposure should be evident at the functional level.…”

Differential responses to maternal diabetes in embryo and visceral yolk sac

“…RNA extraction, library preparation, and RNA sequencing RNA was extracted using the Trizol protocol (Kappen et al, 2022), and quality was assessed on an Agilent Bioanalyzer 2,100 (Agilent Technologies, Santa Clara, CA); only samples with an RNA Integrity Index larger than 7 were used in subsequent experiments. 50 ng of total RNA per sample were used to construct Illuminacompatible RNA sequencing libraries, applying the Lexogen 3′-Quant-Seq method, and utilizing Unique Molecular Identifiers (UMI) in order to guard against PCR-based artefacts when counting sequencing reads (reagents and protocol from Lexogen, Inc., Greenland, NH).…”