319 Single-cell profiling reveals impaired mitochondrial function in mature β-cell population from growth restricted sheep fetuses.

Abstract Placental insufficiency causes fetal growth restriction (FGR) and impairs insulin secretion. β-cell mass and pancreatic insulin content are reduced in the FGR fetus. In prior analysis, islet cell heterogeneity was determined with single-cell RNA sequencing (scRNA-seq) approaches and identified alterations in β-cell maturational progression within fetal sheep islets. Notably, FGR fetuses exhibited a higher population of mature β-cells than controls. Our aim was to elucidate molecular mechanisms that contribute to mature β-cell dysfunction in FGR fetuses. Fetuses with placental insufficiency-induced FGR were generated by exposing pregnant ewes to environmental hyperthermia during mid-gestation. FGR fetuses (n=5, 2 males, 3 females) were compared to control fetuses from thermoneutral ewes (n=4, 2 females, 2 males). Pancreatic tissue was collected, and β-cell RNA was isolated. ScRNA-seq was used to generate transcriptional profiles of β-cells from control and FGR fetal islets. Pseudo-bulk samples were created by filtering mature β-cells per data set. Differentially expressed genes (DEGs) were obtained using DESeq2 (Padj< 0.2), and functional pathway modeling was conducted with KOBAS. Chromogenic RNAscope assays were performed for maturational genes. FGR fetuses had significantly lower body weights than controls (1.2±0.1 vs. 3.3±0.5 kg, P< 0.01). A total of 19,376 genes were expressed across 15,400 and 12,220 cells from control and FGR fetuses, respectively. Sixteen distinct cell types were identified using a two-dimensional UMAP. Insulin-expressing cells were further analyzed for oxidative metabolism enzyme expression to distinguish immature and mature β-cell subpopulations, with MDH2 and ATP6V0B expression validating β-cell maturation. Mature β-cells comprised 17% of the FGR islet cell population, compared to 6% in control islets. A total of 259 DEGs were identified. Enriched pathways like cellular stress response, protein digestion and absorption, oxidative phosphorylation, extracellular matrix-receptor interaction, thermogenesis, PI3K-AKT signaling, and metabolic pathways were downregulated in mature β-cells from FGR fetuses compared to controls. Only proteasome pathway was found upregulated in FGR mature β-cells compared to controls. Key genes involved in mitochondrial respiratory chain were found downregulated in FGR mature β-cells, including COX2, COX3, CYTB, ATP6V0A1, and ND3. Downregulation of these genes suggests impaired mitochondrial respiration and ATP production, which are critical for β-cell function and insulin secretion. Given that mature β-cells rely on oxidative phosphorylation for glucose-stimulated insulin secretion, these results indicate reduced insulin secretory capacity in FGR mature β-cells. Additionally, disruptions in cellular stress responses and extracellular matrix remodeling can impair β-cells maturation, function, and insulin capacity, all of which may increase the risk for developing metabolic complications later in life.