Toll‐like receptor 4 (TLR4) and TLR2 were shown to be activated by saturated fatty acids (SFAs) but inhibited by docosahexaenoic acid (DHA). However, one report (ATVB 11:1944, 2009) suggested that SFA‐induced TLR activation in cell culture systems is due to contaminants in BSA used for conjugating fatty acids. This report casted doubt about proinflammatory effects of SFAs. Our studies herein demonstrate that sodium palmitate (C16:0) or laurate (C12:0) without BSA conjugation induced phosphorylation of IκBα, JNK, ERK, and NFκB p65 and TLR target gene expression in THP1 monocytes or RAW264.7 macrophages, respectively when cultured in low FBS (0.25%) medium. C12:0 induced NFκB activation through TLR2 dimerized with TLR1 or TLR6, and through TLR4. Since BSA was not used in these experiments, contaminants in BSA have no relevance. Unlike suspension cells (THP‐1), BSA conjugation is required for C16:0 to induce TLR target gene expression in adherent cells (RAW264.7). BSA‐conjugated C16:0 transactivated TLR2 dimerized with TLR1 or TLR6, and through TLR4 as seen with C12:0. These results and additional studies with LPS sequester polymixin B and MyD88−/− macrophages indicated that SFA‐induced activation of TLR2 or TLR4 is a fatty acid‐specific effect, but not due to contaminants in BSA or fatty acid preparations. (USDA‐ARS‐WHNRC Program Funds and NIHDK 064007)

Incomplete β-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete β-oxidation) are elevated in T2DM and insulin resistance. In a previous study, we reported that mixed d,l isomers of C12- or C14-carnitine induced an NF-κB-luciferase reporter gene in RAW 264.7 cells, suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant l-acylcarnitines activate classical proinflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of cyclooxygenase-2 in a chain length-dependent manner in RAW 264.7 cells. l-C14 carnitine (5–25 μM), used as a representative acylcarnitine, stimulated the expression and secretion of proinflammatory cytokines in a dose-dependent manner. Furthermore, l-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many proinflammatory signaling pathways including PRRs. Knockdown of MyD88, a key cofactor in PRR signaling and inflammation, blunted the proinflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, l-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking Toll-like receptors (TLR)2 and -4, and did not activate reporter constructs in TLR overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified.