Interaction of palmitate and LPS regulates cytokine expression and apoptosis through sphingolipids in human retinal microvascular endothelial cells

Studies have implicated saturated fatty acid (SFA) and lipopolysaccharide (LPS) in diabetic retinopathy. Since type 2 diabetes is associated with increases in both SFA and LPS in circulation, we investigated how SFA interacts with LPS to regulate proinflammatory cytokine expression and apoptosis in human retinal microvascular endothelial cells (HRMVECs) and the underlying mechanisms. HRMVECs were challenged with palmitate, a major SFA, LPS or palmitate plus LPS and the expression of proinflammatory cytokines were quantified using real-time PCR and enzyme-linked immunosorbent assay. The interaction between palmitate and LPS on inflammatory signaling and sphingolipid metabolism was demonstrated by immunoblotting and lipidomic analysis, respectively. The effect of palmitate and LPS on apoptosis was also studied by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and histone-associated DNA fragment assays. Results showed that palmitate robustly stimulated the expression of proinflammatory cytokines including interleukin (IL)-6 and IL-1 beta, and the combination of palmitate and LPS further upregulated the proinflammatory cytokines by cooperatively stimulating inflammatory signaling pathways. Results also showed that while palmitate stimulated ceramide (CER) production via CER de novo synthesis and sphingomyelin (SM) hydrolysis, addition of LPS further increased CER de novo synthesis, but not SM hydrolysis. The involvement of sphingolipids in the cooperative stimulation by palmitate and LPS on cytokine expression was indicated by the findings that the inhibitor of CER de novo synthesis or SM hydrolysis attenuated the stimulation of IL-6 expression by palmitate and LPS. In addition, our study showed that fatty acid receptors GPR40 and CD36 were involved in the IL-6 upregulation by palmitate and LPS. Furthermore, palmitate induced apoptosis via CER production, but addition of LPS did not further increase apoptosis. Taken together, this study showed that palmitate interacted with LPS to upregulate cytokine expression via free fatty acid receptor-mediated inflammatory signaling and sphingolipid metabolism in HRMVECs. In contrast, the interaction between palmitate and LPS did not further increase apoptosis.