OBJECTIVE: Previous studies demonstrated that obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that they might represent an important source of inflammation. Using an in vitro coculture system composed of 3T3-L1 adipocytes and RAW264 macrophages, we previously demonstrated that saturated fatty acids (FAs) and tumor necrosis factor (TNF)-alpha derived from adipocytes and macrophages, respectively, play a major role in the coculture-induced inflammatory changes. METHODS AND RESULTS: Coculture of adipocytes and macrophages resulted in the activation of nuclear factor-kappaB (NF-kappaB), a primary regulator of inflammatory responses, in both cell types. Pharmacological inhibition of NF-kappaB markedly suppressed the coculture-induced production of proinflammatory cytokines and adipocyte lipolysis. Peritoneal macrophages obtained from Toll-like receptor 4 (TLR4) mutant mice exhibited marked attenuation of TNFalpha production in response to saturated FAs. Notably, coculture of hypertrophied adipocytes and TLR4-mutant macrophages resulted in marked inhibition of proinflammatory cytokine production and adipocyte lipolysis. We also observed that endogenous FAs, which are released from adipocytes via the beta3-adrenergic stimulation, resulted in the activation of the TLR4/NF-kappaB pathway. CONCLUSIONS: These findings suggest that saturated FAs, which are released in large quantities from hypertrophied adipocytes via the macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for TLR4, thereby inducing the inflammatory changes in both adipocytes and macrophages through NF-kappaB activation.