Plasma acylcarnitine profiles suggest incomplete long-chain fatty acid beta-oxidation and altered tricarboxylic acid cycle activity in type 2 diabetic African-American women

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, 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.

Figures

FIGURE 1

Evidence for alteration of acetylcarnitine and propionylcarnitine metabolism with increasing severity of diabetes and association with markers of incomplete fatty acid β-oxidation. (A) Significant positive correlation between fasting blood HbA1c and plasma acetylcarnitine concentration (Pearson correlation statistic, n = 56). (B) Association between the relative abundance of plasma acetylcarnitine (left axis, circles) and plasma propionylcarnitine (right axis, triangles) to increasing plasma acylcarnitine concentrations across all participants; diabetics (open circles, black triangles) and nondiabetics (gray symbols) are illustrated (2 diabetic outliers with relative levels of 40% and 60% of summed acylcarnitines are not shown). Best-fit lines were described by the equation: y = a × Ln(x) + b.

FIGURE 2

Activity of NFκB is stimulated by MCFA-carn in RAW264.7 murine monocyte cells. Cells transfected with a (2×)-NF-κB-Luc containing 2 copies of the NFκB consensus-binding site were treated for 15 h with 20 μmol/L MCFA (C12:0 and C14:0), 20 μmol/L dl-carnitine alone, or 20 μmol/L dl-MCFA-carn (C12-carn and C14-carn). Relative luciferase activity (RLA) changes relative to vehicle control RLA are illustrated, with 5 μg/L LPS serving as a positive control. Results were derived from 3 (C12 studies; n = 9) to ≥3 (C14 studies; n = 21) independent cell culture experiments. Values are means ± SEM. **Different from vehicle-treated control, P < 0.01.

FIGURE 3

Working model of tissue metabolism in T2DM and potential links between the TCA cycle, fatty acid metabolism, acylcarnitines, and insulin resistance. Considering results from the current experiments and literature reports, a unifying model was devised to illustrate metabolic and inflammatory events that may take place under diabetic conditions. This hypothetical paradigm proposes that in type 2 diabetic tissues, there is: 1) an inadequate capacity of the TCA relative to fuel delivery, due in part to reduced anaplerotic propionyl-CoA pools reflected in lower propionylcarnitine levels; 2) mismatched acetyl-CoA generation vs. entry into the TCA cycle, leading to accumulation of mitochondrial acetyl-CoA and hence increased acetylcarnitine generation via carnitine acyltransferase (CAT) activity (specifically, carnitine acetyltransferase); 3) incomplete LCFA catabolism and accumulation of chain-shortened acyl-CoA moieties that serve as substrates for CAT, with subsequent generation of medium-chain acylcarnitines. Thioesterases (THEA) might also act on acyl-CoA to increase free MCFA generation; 4) entry of a fraction of MCFA-carn into the bloodstream/extracellular space surrounding affected tissue; and 5) activation of proinflammatory NFκB through cell-surface TLR or other mechanisms, in turn promoting local or systemic insulin resistance.