No abstract

Results from our previous studies demonstrated that activation of Toll-like receptor 4 (Tlr4), the lipopolysaccharide (LPS) receptor, is sufficient to induce nuclear factor B activation and expression of inducible cyclooxygenase (COX-2) in macrophages. Saturated fatty acids (SFAs) acylated in lipid A moiety of LPS are essential for biological activities of LPS. Thus, we determined whether these fatty acids modulate LPS-induced signaling pathways and COX-2 expression in monocyte/macrophage cells (RAW 264.7). Results show that SFAs, but not unsaturated fatty acids (UFAs), induce nuclear factor B activation and expression of COX-2 and other inflammatory markers. This induction is inhibited by a dominant-negative Tlr4. UFAs inhibit COX-2 expression induced by SFAs, constitutively active Tlr4, or LPS. However, UFAs fail to inhibit COX-2 expression induced by activation of signaling components downstream of Tlr4. Together, these results suggest that both SFA-induced COX-2 expression and its inhibition by UFAs are mediated through a common signaling pathway derived from Tlr4. These results represent a novel mechanism by which fatty acids modulate signaling pathways and target gene expression. Furthermore, these results suggest a possibility that propensity of monocyte/macrophage activation is modulated through Tlr4 by different types of free fatty acids, which in turn can be altered by kinds of dietary fat consumed. Cyclooxygenase (COX;1 prostaglandin endoperoxide (PGH 2 ) synthase) catalyzes the conversion of arachidonic acid to prostaglandin endoperoxide. This is the rate-limiting step in prostaglandin and thromboxane biosynthesis. Two isoforms of COX have been cloned from various animal cells, constitutively expressed COX-1 (1-5) and mitogen-inducible COX-2 (6 -11). Numerous studies have demonstrated that the levels of prostaglandins in various tumors, or the tumor's biosynthetic capacity of prostaglandins, are greater when compared with normal tissues (12-16). Recently, it has been shown that the inducible form of COX is overexpressed in sites of inflammation and in many types of tumor tissues (17)(18)(19)(20). Overexpression of COX-2 in tumor tissues occurs in both tumor cells and stromal cells including macrophages (21). What causes the overexpression of COX-2 in such pathological states is not clearly understood. COX-2 belongs to a family of immediate early response genes that do not require precedent protein synthesis for their expression (22). Therefore, elucidating the signaling pathways leading to the expression of COX-2 is a key to understanding why COX-2 is overexpressed in such pathological states and can provide critical information for identifying potential targets of modulation by pharmacological and dietary factors.COX-2 expression is induced by various mitogenic stimuli in different cell types (6,9,11,23). The cis-acting NFB element is present in the 5Ј-flanking regions of COX-2 genes of different species (24,25). Results from our previous studies demonstrated that the activation of NFB is required t...

Inefficient muscle long-chain fatty acid (LCFA) combustion is associated with insulin resistance, but molecular links between mitochondrial fat catabolism and insulin action remain controversial. We hypothesized that plasma acylcarnitine profiling would identify distinct metabolite patterns reflective of muscle fat catabolism when comparing individuals bearing a missense G304A uncoupling protein 3 (UCP3 g/a) polymorphism to controls, because UCP3 is predominantly expressed in skeletal muscle and g/a individuals have reduced whole-body fat oxidation. MS analyses of 42 carnitine moieties in plasma samples from fasting type 2 diabetics (n = 44) and nondiabetics (n = 12) with or without the UCP3 g/a polymorphism (n = 28/genotype: 22 diabetic, 6 nondiabetic/genotype) were conducted. Contrary to our hypothesis, genotype had a negligible impact on plasma metabolite patterns. However, a comparison of nondiabetics vs. type 2 diabetics revealed a striking increase in the concentrations of fatty acylcarnitines reflective of incomplete LCFA beta-oxidation in the latter (i.e. summed C10- to C14-carnitine concentrations were approximately 300% of controls; P = 0.004). Across all volunteers (n = 56), acetylcarnitine rose and propionylcarnitine decreased with increasing hemoglobin A1c (r = 0.544, P < 0.0001; and r = -0.308, P < 0.05, respectively) and with increasing total plasma acylcarnitine concentration. In proof-of-concept studies, we made the novel observation that C12-C14 acylcarnitines significantly stimulated nuclear factor kappa-B activity (up to 200% of controls) in RAW264.7 cells. These results are consistent with the working hypothesis that inefficient tissue LCFA beta-oxidation, due in part to a relatively low tricarboxylic acid cycle capacity, increases tissue accumulation of acetyl-CoA and generates chain-shortened acylcarnitine molecules that activate proinflammatory pathways implicated in insulin resistance.