• TLR4 directly interacts with TMEM24, downregulating its expression to inhibit the PI3K/AKT pathway, thereby mediating lipotoxic β-cell dysfunction.
• TMEM24 is identified as a novel target of palmitic acid-induced insulin secretion impairment, linking lipotoxicity to impaired pulsatile insulin release.
• TLR4 knockout in high-fat diet-induced obese rats restores islet function via the TMEM24/PI3K/AKT signaling axis, suggesting a potential therapeutic strategy.
• The study provides mechanistic insight into how metabolic inflammation impairs insulin vesicular secretion, offering new avenues for targeted interventions in type 2 diabetes.