PDK4 is critical for metabolic regulation and progesterone production in bovine granulosa cells†

Non-esterified fatty acids (NEFAs) influence ovarian cell function, but the underlying molecular mechanisms remain incompletely understood. Our previous work showed that NEFA induces lipid accumulation and inhibits glucose uptake in bovine granulosa cells. Here, we investigated the effect of NEFA on energy metabolism and the transcriptome, with a focus on pyruvate dehydrogenase kinase 4 (PDK4). NEFA treatment significantly increased ATP levels and mitochondrial membrane potential. Transcriptome analysis revealed differential expression of 176 genes, with downregulation of steroidogenesis and fatty acid synthesis pathways, and upregulation of apoptosis, fatty acid oxidation, and innate immune responses in NEFA treated cells. Specifically, genes involved in fatty acid oxidation (e.g. CPT1A, CPT1B, HADHA, and SLC25A20) were upregulated, whereas those related to glucose metabolism remained largely unchanged except for the marked upregulation of PDK4. Silencing PDK4 expression induced glucose utilization by upregulating glucose transporters (SLC2A1, SLC2A10) and glycolytic enzymes (GAPDH, ENO1, and LDHA). In contrast, genes associated with fatty acid oxidation (SLC27A1 and CPT1B) showed downregulation upon PDK4 silencing compared with controls, suggesting a metabolic shift favoring glucose oxidation even in the presence of NEFA. We observed that expression of PDK4, CPT1A, and CPT1B was significantly upregulated in large luteal cells compared to granulosa cells. PDK4 knockdown significantly downregulated steroidogenic genes (STAR, HSD3B1, and CYP11A1) and decreased progesterone production, suggesting that increased expression of PDK4 in luteal cells may supports steroidogenesis. Together, these findings identify PDK4 as a critical regulator of metabolic flexibility and progesterone production in granulosa cells.