Palmitate drives mitochondrial and ER stress through disruption of the CD73-Adenosine axis.

Metabolic disorders associated with elevated saturated fatty acids are linked to chronic inflammatory diseases, including periodontitis, yet the mechanisms connecting lipotoxic stress to gingival inflammation remain unclear. This study investigated how palmitate-induced metabolic stress affects purinergic signaling, mitochondrial function, and endoplasmic reticulum (ER) stress in murine gingival fibroblasts (mGF), and whether adenosine modulates these effects. mGF were treated with BSA control, palmitate, IL-1β, or palmitate plus IL-1β, followed by bulk RNA sequencing, Seahorse metabolic analysis, biochemical assays, and transmission electron microscopy. Palmitate suppressed expression of key adenosine-generating ectoenzymes and purinergic signaling genes, including Cd73 (Nt5e), Cd39 (Entpd1), Adk, Ada, and adenosine receptors. Concurrently, palmitate amplified IL-1β-induced inflammatory mediators such as Cxcl1, Cxcl2, Cxcl5, Ccl2, and Il6. Gene ontology analysis demonstrated enrichment of pathways related to innate immune activation, oxidative stress, mitochondrial dysfunction, ER stress, and purine metabolism. Palmitate also induced intracellular lipid accumulation and mitochondrial dysfunction, evidenced by reduced NAD+/NADH ratio, increased mitochondrial reactive oxygen species (ROS), elevated protein oxidation, and increased proton leak despite enhanced electron transport chain protein expression. Ultrastructural analyses revealed swollen mitochondria, ER expansion, and increased ER-mitochondrial associations. Mechanistically, palmitate activated the Perk-eIF2α-Atf4 ER stress pathway, increasing phosphorylation of Perk and eIF2α and elevating Atf4 expression. Extracellular adenosine attenuated mitochondrial ROS accumulation, reversed Perk and Atf4 activation, improved mitochondrial respiration, and preserved ER and mitochondrial ultrastructure. Palmitate disrupts the Cd73-adenosine axis while promoting mitochondrial dysfunction, oxidative stress, and Perk-mediated ER stress in gingival fibroblasts. Adenosine signaling protects against lipotoxic-induced ER stress, highlighting the Cd73-adenosine pathway as a potential therapeutic target in metabolically driven periodontal inflammation.