Palmitic Acid Stimulates A Delayed But Extremely High Expression Of Il-6 And Also Enhances Lps Signaling Via Sphingolipid Metabolism In Human Aortic Endothelial Cells

It is known that both saturated fatty acids (SFAs) and lipopolysaccharide (LPS) boost host chronic inflammatory response that contributes to inflammation-related diseases such as diabetes and atherosclerosis. However, it remains unclear how SFAs interact with LPS to promote inflammation. In this study, we compared the effects of LPS, palmitic acid (PA), a most abundant SFA, and the combination of LPS and PA on the expression of interleukin (IL)-6, a key pro-inflammatory cytokine, in human aortic endothelial cells (HAECs). Results showed that while LPS induced a quick but brief IL-6 mRNA expression that peaked at 12 h, PA stimulated a delayed but extremely high expression of IL-6 mRNA (22-fold of that stimulated by LPS) at 36 h (IL-6 mRNA/house-keeping gene mRNA ratios: control-0.21; LPS-0.4; PA-8.68). Results also showed that the combination of LPS and PA further increased IL-6 mRNA at 36 h (IL-6 mRNA/house-keeping gene mRNA ratios: PA-8.68; LPS plus PA-14.23). Consistently, quantification of IL-6 protein in culture medium showed that PA stimulated more IL-6 secretion than LPS and PA in the combination of LPS led to a much greater IL-6 secretion at 36 h (control-269; PA-2485; LPS-1258; LPS plus PA-5811 pg/ml). Since PA is known to affect ceramide biosynthesis, we determine if sphingolipid metabolism is involved in IL-6 upregulation by LPS, PA or LPS plus PA. Results showed that PA significantly increased sphingomyelin (SM) hydrolysis and PA in combination with LPS further increased it (cellular SM levels: control-18.5; LPS-16.5; PA-13.6; LPS plus PA-10.7 pmol/ng phosphate). Increased SM hydrolysis was associated with an increase in ceramide, a bioactive sphingolipid known to stimulate inflammatory cytokines. Moreover, using pharmacological inhibitor and RNA interfering, we found that neutral sphingomyelinase (nSMase), but not acid SMase, was involved in the SM hydrolysis. Finally, we found that inhibition of SM hydrolysis by nSMase inhibitor or nSMase siRNA markedly inhibited IL-6 expression stimulated by LPS, PA or LPS plus PA. In conclusion, this study demonstrated that PA not only stimulated a delayed but extremely high expression of IL-6, but also enhanced the effect of LPS in HAEC, and sphingolipid metabolism played a crucial role in the PA action.