Technical Abstract: 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, since UCP3 is predominantly expressed in skeletal muscle and g/a individuals have reduced whole-body fat oxidation. Mass-spectroscopy analyses of 42 acylcarnitine moieties in plasma samples from overnight-fasted type 2 diabetics (n=44) and non-diabetics (n=12) with or without the UCP3 g/a polymorphism (n=28/genotype: 22 diabetic, 6 non-diabetic/genotype) were conducted. Contrary to our hypothesis, genotype had negligible impact on plasma metabolite patterns. However, a comparison of non-diabetics vs. type 2 diabetics revealed a striking increase in the concentrations of fatty acylcarnitines reflective of incomplete LCFA ß-oxidation in the latter (i.e., summed C10- to C14-carnitine concentrations were ~300% of controls, p=0.004). Across all volunteers (n=56), acetylcarnitine rose and propionylcarnitine decreased with increasing hemoglobin A1c (HbA1c; 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 acylarnitines significantly stimulated NF'B activity (up to 200% of controls) in RAW264.7 cells. These results are consistent with the working hypothesis that inefficient tissue LCFA ß-oxidation, due in part to a relatively low tricarboxylic acid (TCA) cycle capacity, increases tissue accumulation of acetyl-CoA and generates chain-shortened acylcarnitine molecules that activate pro-inflammatory pathways implicated in insulin resistance.