Multi-Omics Analyses Reveal Expression Remodeling of Key Glycolytic-Lactate Metabolic Nodes in the Pituitary of High-Altitude Sheep

Background: Most studies of high-altitude adaptation in animals have focused on the heart, lung, blood, and skeletal muscle, whereas systematic evidence regarding metabolism-related expression changes in the pituitary remains limited. The pituitary is an energy-demanding neuroendocrine organ characterized by active glucose utilization and lactate production. However, multi-omics changes associated with glycolysis–lactate metabolism in the pituitary of high-altitude sheep remain undefined. Methods: Pituitary tissues from adult female sheep living at high altitude were compared with those from adult female sheep living at low altitude (n = 3 per group). Transcriptomic differences were profiled by RNA sequencing, and proteomic differences were characterized by data-independent acquisition (DIA) mass spectrometry (MS). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, gene set enrichment analysis, and integrated transcriptomic–proteomic analyses were used to identify representative candidates, which were further validated by reverse transcription quantitative polymerase chain reaction and western blotting. Results: RNA sequencing identified 1222 differentially expressed genes, including 690 upregulated and 532 downregulated genes, whereas DIA-MS identified 2046 differentially expressed proteins, including 1096 upregulated and 950 downregulated proteins. Functional analyses indicated enrichment of glycolysis, lactate metabolism, pyruvate metabolism, the HIF-1 signaling pathway, and oxidative phosphorylation. Gene set enrichment analysis showed significant positive enrichment of oxidative phosphorylation in the high-altitude group, whereas glycolysis/gluconeogenesis showed a positive trend without meeting the false discovery rate threshold. Integrated transcriptomic–proteomic analysis identified hexokinase 2 (HK2), enolase 2 (ENO2), and lactate dehydrogenase A (LDHA) as concordantly upregulated at both the transcript and protein levels (q-value < 0.05). Reverse transcription quantitative PCR (RT-qPCR) showed significant upregulation of HK2, ENO2, and LDHA (all p < 0.001), while western blotting confirmed increased protein expression, with HK2 reaching p < 0.001 and ENO2 and LDHA reaching p < 0.01. Conclusions: HK2, ENO2, and LDHA are stably upregulated in the pituitary of high-altitude sheep, indicating remodeling of key expression nodes associated with glycolysis–lactate metabolism. These findings provide multi-omics evidence for high-altitude-associated remodeling of selected metabolic expression nodes in the sheep pituitary.