Sex‐Specific Anti‐Inflammatory Effects of Alternate Day Fasting via TLR4 and Leptin Signaling in Middle‐Aged Rats

Alternate-day fasting (ADF) may exert age-related anti-inflammatory effects by modulating key metabolic and immune pathways. This study aimed to investigate the sex-specific effects of ADF on inflammation in middle-aged male and female rats and focused on the Toll-like receptor 4 (TLR4) and leptin signaling pathways. A total of 32 rats (16 males, 16 females; 14 months old) were assigned to four groups (n = 8): ad libitum-fed and ADF groups for each sex. The ADF protocol consisted of 24-h fasting every other day for 2 months. Serum and hypothalamic samples were analyzed via ELISA to measure triglyceride, total cholesterol, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), suppressor of cytokine signaling 3 (SOCS3), leptin, and ObRb levels. TLR4 gene expression in the brain and liver was assessed via qRT-PCR. Two months of ADF significantly reduced body weight and retroperitoneal fat mass compared with those of the control groups (p < 0.001). Cumulative food intake and fasting glucose levels decreased in both sexes following ADF (p < 0.001), and serum triglyceride, total cholesterol, TNF-α, and IL-6 levels (p < 0.001) were significantly reduced. Hypothalamic SOCS3 and leptin levels decreased, whereas ObRb expression increased (p < 0.05). Additionally, TLR4 gene expression was significantly downregulated in both tissues (p < 0.001). These findings suggest that ADF is associated with reduced age-related inflammation and alterations in the TLR4 and leptin signaling pathways. The observed downregulation of SOCS3 and upregulation of ObRb indicate enhanced leptin sensitivity, highlighting ADF as a promising strategy to counteract sex-specific aging-related inflammation and leptin resistance.