Carnitine acetyltransferase (CrAT) is a mitochondrial matrix
enzyme that catalyzes the interconversion of
acetyl-CoA and
acetylcarnitine. Emerging evidence suggests that this
enzyme functions as a positive regulator of total body
glucose tolerance and muscle activity of
pyruvate dehydrogenase (PDH), a mitochondrial
enzyme complex that promotes
glucose oxidation and is feedback inhibited by
acetyl-CoA. Here, we used tandem mass spectrometry-based metabolic profiling to identify a negative relationship between CrAT activity and muscle content of
lipid intermediates. CrAT specific activity was diminished in muscles from obese and diabetic rodents despite increased
protein abundance. This reduction in
enzyme activity was accompanied by muscle accumulation of long-chain acylcarnitines (LCACs) and acyl-CoAs and a decline in the
acetylcarnitine/
acetyl-CoA ratio. In vitro assays demonstrated that
palmitoyl-CoA acts as a direct mixed-model inhibitor of CrAT. Similarly, in primary human myocytes grown in culture, nutritional and genetic manipulations that promoted mitochondrial influx of
fatty acids resulted in accumulation of LCACs but a pronounced decrease of CrAT-derived short-chain acylcarnitines. These results suggest that
lipid-induced antagonism of CrAT might contribute to decreased PDH activity and
glucose disposal in the context of
obesity and diabetes.