High-resolution respirometry for measurement of fatty acid β-oxidation in brain mitochondria. A comparative study and application in ischemia and aging

Although glucose and ketone bodies are the main energy sources of the brain, fatty acid oxidation (FAO) plays a relevant role in the pathogenesis of central nervous system disorders. Given the low contribution of FAO to brain metabolism, protocols for its assessment must be carefully designed.
High-resolution respirometry protocols were developed to avoid FAO overestimation by malate-linked anaplerotic activity in brain mitochondria. We compared octanoylcarnitine (medium-chain) and palmitoylcarnitine (long-chain acylcarnitine) as substrates of β-oxidation in the brain. The capacity of FA oxidative phosphorylation (F-OXPHOS) with palmitoylcarnitine was up to 4 times higher than respiration with octanoylcarnitine. The optimal concentration of palmitoylcarnitine was 10 μM which corresponds to the total concentration of long-chain acylcarnitines in the brain. Higher concentrations of palmitoylcarnitine inhibited respiration, showing that optimization is essential to avoid inhibitory concentrations of this substrate. Maximal respiration with octanoylcarnitine was reached at 20 μM, however, this concentration exceeds medium-chain acylcarnitine concentrations in the brain 15 times.
Brain mitochondrial F-OXPHOS capacity was higher in mice and rats compared to Drosophila. However, compared to other respiratory rates, FAO-linked respiration had a higher contribution in Drosophila brains. F-OXPHOS capacity was highest in mouse cerebellum, intermediate in the cortex, prefrontal cortex, and hypothalamus, and hardly detectable in the hippocampus.
In a rat model of endothelin-1-induced stroke, OXPHOS capacities showed a trend to decline which was not limited to F-OXPHOS, whereas medium- and long-chain acylcarnitine levels increased. In aged rats, F-OXPHOS capacity declined more in comparison to other OXPHOS capacities, being 2-fold lower than in the younger control group, while concentrations of long-chain acylcarnitines were 2-fold higher.
These data indicate that, although FAO is not a dominant pathway in brain energy metabolism, it plays important roles, such as avoiding the accumulation of long-chain acylcarnitines. Further studies of fatty acid oxidation deserve attention in brain bioenergetics and its relations to physiology and pathology.