Offspring of diabetic mothers are susceptible to developing type 2 diabetes due to pancreatic islet dysfunction. However, the initiating molecular pathways leading to offspring pancreatic islet dysfunction are unknown. We hypothesized that maternal hyperglycemia alters offspring pancreatic islet transcriptome and negatively impacts offspring islet function. We employed an infusion model capable of inducing localized hyperglycemia in fetal rats residing in the left uterine horn, thus avoiding other factors involved in programming offspring pancreatic islet health. While maintaining euglycemia in maternal dams and right uterine horn control fetuses, hyperglycemic fetuses in the left uterine horn had higher serum insulin and pancreatic beta cell area. Upon completing infusion from GD20 to 22, RNA sequencing was performed on GD22 islets to identify the hyperglycemia-induced altered gene expression. Ingenuity pathway analysis of the altered transcriptome found that diabetes mellitus and inflammation/cell death pathways were enriched. Interestingly, the downregulated genes modulate more diverse biological processes, which includes responses to stimuli and developmental processes. Next, we performed ex and in vivo studies to evaluate islet cell viability and insulin secretory function in weanling and adult offspring. Pancreatic islets of weanlings exposed to late gestation hyperglycemia had decreased cell viability in basal state and glucose-induced insulin secretion. Lastly, adult offspring exposed to in utero hyperglycemia also exhibited glucose intolerance and insulin secretory dysfunction. Together, our results demonstrate that late gestational hyperglycemia alters the fetal pancreatic islet transcriptome and increases offspring susceptibility to developing pancreatic islet dysfunction.
Supplemental Figure 1: GD22 fetal pancreatic sections (n=3 pups/group from 3 mothers of independent infusions) were stained with the aforementioned anti-insulin Ab and DAPI, and the number of nuclei (DAPI) were counted and normalized to 1000 μm2 insulin-positive area.
Supplemental Figure 2: 3 GD20 pregnant dams underwent the exact same surgery with saline infused into left uterine artery (SAL). Male pups were cross-fostered and GTT was performed on postnatal day 21 as described in the methods section. The glucose levels between 2 groups are analyzed using the same statistical approach as described in manuscript (two-way ANOVA tests followed by Bonferroni multiple comparison tests)
Supplemental Figure 3: Measurement of serum (A) Interferon-gamma, (B) Tnf-alpha, (C) ll-1B, (D) IL-17, and (E) Cxcl10 showing no difference between 2 groups. Each symbol represents one replicate with solid line connecting paired group (n=3-5/group, from three to five mothers). Serum was collected from fetal, neonatal, and adult offspring of Con and HG, then measured using Milliplex Rat Cytokine/Chemokine Magnet­ic Bead Panel - Immunology Multiplex Assay (RECYTMAG-65K, Millipore Sigma, MA) by Indiana University Multiplex Analysis Core. This assay is designed to simultaneously quantify selected rat cyto­kines. The kit contains spectrally distinct antibody-immobilized beads, cytokine standard cocktail, streptavidin-phycoerythrin, assay buffer, wash buffer, serum matrix, and microtitre filter plate. Following the manufacturer's recommendation, 25 ul of samples were diluted (1 :2) and processed, then analyzed using Bio-Plex 200 System with High Throughput Fluidics (HTF) Multiplex Assay Array System (Bio-Rad Laboratories, Hercules, CA)> All the samples were run in duplicate. The detection limits for the measured cytokines are as follow: IFN-Gamma 6.2 pg/ml, TNF-Alpha 1.9 pg/ml, IL-1 B 2.8 pg/ml, IL-17 2.3 pg/ml, Cxcl10 1.4 pg/ml).
Supp Table 1: List of Differentially expressed genes in HG GD22 islets
Supp Table 2: Pathways enriched by IPA
Supp Table 3: EnrichR Analysis of downregulated genes