402 - Multigenerational consequences of maternal high fat diet and gestational diabetes (GDM) at the preimplantation embryo stage

Publication Number: 402.6007

Srikrishna P Reddy, University of South Dakota, Sanford School of Medicine, Sioux Falls, SD, United States; Abigail K Klein, University of South Dakota, Sanford School of Medicine, Sioux Falls, SD, United States; Benjamin Derenge, Sanford Children's Hospital, Sioux Falls, SD, United States; Tricia D. Larsen, Sanford Research, Sioux Falls, SD, United States; Prathapan Ayyappan, Sanford Children's Hospital, Sioux Falls, SD, United States; Michael S. Kareta, University of South Dakota, Sanford School of Medicine, Sioux Falls, SD, United States; Michelle L. Baack, University of South Dakota, Sanford School of Medicine, Sioux Falls, SD, United States

Background: Diabetic pregnancy increases the risk of cardiometabolic disease in offspring across the lifespan, but mechanisms and timing are not understood. We used rat models to show that a maternal high fat (HF) diet and late-gestation diabetes incite mitochondrial dysfunction, oxidative stress and cardiomyopathy in 1st generation (F1) progeny. Because HF diet can cause mitochondrial dysfunction in maternal & fetal oocytes and mitochondria are maternally inherited, we hypothesize that mitochondria-mediated heart disease is preprogrammed at the embryonic stage and can be inherited across generations

Objective: Determine the effects of maternal diabetes and high-fat diet on 2nd and 3rd generation preimplantation embryos.

Design/Methods: Female rats were fed a control (CD) or HF diet and bred with CD-fed males. On embryonic day (E)14, a subset of HF-fed rats was given streptozocin (STZ) to induce diabetes which was treated with insulin. F1 pups were cross-fostered to CD-fed dams, weaned to CD and later bred with CD-fed males to produce 2nd generation (F2) pups and embryos (F2e). F2 pups were bred with CD-fed males to produce 3rd generation embryos (F3e). Embryos were assessed by live-cell imaging and single-cell RNA sequencing (scRNA-seq) using 10X Genomics. Image analysis was done using ImageJ. Differentially expressed genes (DEG) were identified using Seurat package in R and analyzed using the MAST test. Significance was set at p< 0.05 and log2 fold change > |1.25|. Group and generational differences were compared using Student’s t-test with significance set at p≤ 0.05.

Results: Although F2e had no difference in expression of mitochondrial genes compared to controls (Ce), F3e, which had no direct exposure, had higher mitochondrial gene expression compared to both Ce and F2e (p < 0.05) (Fig1). Increased mitochondria quantity was validated by more staining compared to Ce (p < 0.01) (Fig 1). F2e had increased expression of genes activated during oxidative stress (p < 0.001) and more staining of reactive oxygen species (ROS) (p < 0.01) compared to Ce (Fig 2). Both expression of oxidative stress genes and ROS staining decreased to Ce levels (p < 0.05) in F3e (Fig 2). Other DEGs in F2e which returned to Ce levels in F3e included upregulated genes involved in RNA splicing and regulation of DNA recombination (Fig 3).

Conclusion(s): Diet and diabetic pregnancy can program the early embryonic transcriptome with consequences for next generations even if a healthy diet is maintained throughout life. Our findings suggest that exposing primordial oocytes to diabetic fuels is detrimental, but thankfully is reversible in unexposed F3e.

Comparison of mitochondrial gene expression between control and HFSTZ F2 and F3 embryos
F3e, which had no direct exposure, had higher mitochondrial gene expression compared to both Ce and F2e (validated by increased staining)

Comparison of genes activated during oxidative stress between control and HFSTZ F2 and F3 embryos
F2e had increased expression of genes activated during oxidative stress and more staining of reactive oxygen species compared to Ce. Both expression of oxidative stress genes and ROS staining decreased to Ce levels in F3e

Pathway analysis showing comparison of DEGs between Control and F2/F3 embryos
Figure showing upregulation of genes involved in RNA splicing, regulation of DNA recombination in F2e which returned to baseline in F3e