Abstract
Background/AimIncomplete or limited long-chain fatty acid (LCFA) combustion in skeletal muscle has been associated with insulin resistance. Signals that are responsive to shifts in LCFA β-oxidation rate or degree of intramitochondrial catabolism are hypothesized to regulate second messenger systems downstream of the insulin receptor. Recent evidence supports a causal link between mitochondrial LCFA combustion in skeletal muscle and insulin resistance. We have used unbiased metabolite profiling of mouse muscle mitochondria with the aim of identifying candidate metabolites within or effluxed from mitochondria and that are shifted with LCFA combustion rate.Methodology/Principal FindingsLarge-scale unbiased metabolomics analysis was performed using GC/TOF-MS on buffer and mitochondrial matrix fractions obtained prior to and after 20 min of palmitate catabolism (n = 7 mice/condition). Three palmitate concentrations (2, 9 and 19 µM; corresponding to low, intermediate and high oxidation rates) and 9 µM palmitate plus tricarboxylic acid (TCA) cycle and electron transport chain inhibitors were each tested and compared to zero palmitate control incubations. Paired comparisons of the 0 and 20 min samples were made by Student's t-test. False discovery rate were estimated and Type I error rates assigned. Major metabolite groups were organic acids, amines and amino acids, free fatty acids and sugar phosphates. Palmitate oxidation was associated with unique profiles of metabolites, a subset of which correlated to palmitate oxidation rate. In particular, palmitate oxidation rate was associated with distinct changes in the levels of TCA cycle intermediates within and effluxed from mitochondria.Conclusions/SignificanceThis proof-of-principle study establishes that large-scale metabolomics methods can be applied to organelle-level models to discover metabolite patterns reflective of LCFA combustion, which may lead to identification of molecules linking muscle fat metabolism and insulin signaling. Our results suggest that future studies should focus on the fate of effluxed TCA cycle intermediates and on mechanisms ensuring their replenishment during LCFA metabolism in skeletal muscle.
Highlights
Long-chain fatty acids (LCFA) are a crucial energy source in mammalian peripheral tissues, including skeletal muscle
Our results suggest that future studies should focus on the fate of effluxed tricarboxylic acid cycle (TCA) cycle intermediates and on mechanisms ensuring their replenishment during LCFA metabolism in skeletal muscle
Cellular disposal of FA is associated with reduced insulin-mediated glucose uptake [2], and a role for cycle flux [22]; in rodents, such ‘mitochondrial FA overload’ was reflected in higher incomplete oxidation, and in increased serum and muscle levels of acylcarnitines [22]
Summary
Long-chain fatty acids (LCFA) are a crucial energy source in mammalian peripheral tissues, including skeletal muscle. Individuals with T2DM have lower skeletal muscle FAO rates in the fasted state [9,10], as well as decreased subsarcolemmal mitochondrial content [11], decreased expression of oxidative phosphorylation genes [12,13], and decreased maximal NADHlinked phosphorylating respiration in isolated mitochondria [14,15]. Overexpression of carnitine palmitoyltransferase 1, a key controlling enzyme in FAO, at physiologic levels improved insulin sensitivity [18]. These findings have suggested that impaired FAO (and mitochondrial function) promotes insulin resistance. Improved insulin sensitivity was correlated with lower FAO rate despite higher intramuscular long-chain acyl-CoA species [22]. It has been proposed that insulin resistance is associated with excessive flux through b-oxidation relative to tricarboxylic acid cycle (TCA) capacity [22,24], a phenomenon termed mitochondrial FA overload [22]
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