AMP-activated protein kinase activators have compound and concentration-specific effects on brain metabolism.

AMP-activated protein kinase (AMPK) is a key sensor of energy balance playing important roles in the balancing of anabolic and catabolic activities. The high energy demands of the brain and its limited capacity to store energy indicate that AMPK may play a significant role in brain metabolism. Here, we activated AMPK in guinea pig cortical tissue slices, both directly with A769662 and PF 06409577 and indirectly with AICAR and metformin. We studied the resultant metabolism of [1-13 C]glucose and [1,2-13 C]acetate using NMR spectroscopy. We found distinct activator concentration-dependent effects on metabolism, which ranged from decreased metabolic pool sizes at EC50 activator concentrations with no expected stimulation in glycolytic flux to increased aerobic glycolysis and decreased pyruvate metabolism with certain activators. Further, activation with direct versus indirect activators produced distinct metabolic outcomes at both low (EC50 ) and higher (EC50 × 10) concentrations. Specific direct activation of ß1-containing AMPK isoforms with PF 06409577 resulted in increased Krebs cycle activity, restoring pyruvate metabolism while A769662 increased lactate and alanine production, as well as labelling of citrate and glutamine. These results reveal a complex metabolic response to AMPK activators in brain beyond increased aerobic glycolysis and indicate that further research is warranted into their concentration- and mechanism-dependent impact.

Correction to: L-Aspartate, L-Ornithine and L-Ornithine-L-Aspartate (LOLA) and Their Impact on Brain Energy Metabolism.

The original version of this published article, the bottom right hand panels of Figs. 3-6 were labelled as "Isotopomers formed from [1-13C]D-glucose". This is incorrect and should read "Isotopomers formed from [1,2-13C]acetate". This has been corrected by publishing this correction article.

L-Aspartate, L-Ornithine and L-Ornithine-L-Aspartate (LOLA) and Their Impact on Brain Energy Metabolism.

L-Ornithine-L-aspartate (LOLA), a crystalline salt, is used primarily in the management of hepatic encephalopathy. The degree to which it might penetrate the brain, and the effects it might have on metabolism in brain are poorly understood. Here, to investigate the effects of LOLA on brain energy metabolism we incubated brain cortical tissue slices from guinea pig (Cavea porcellus) with the constituent amino acids of LOLA, L-ornithine or L-aspartate, as well as LOLA, in the presence of [1-13C]D-glucose and [1,2-13C]acetate; these labelled substrates are useful indicators of brain metabolic activity. L-Ornithine produced significant "sedative" effects on brain slice metabolism, most likely via conversion of ornithine to GABA via the ornithine aminotransferase pathway, while L-aspartate showed concentration-dependent excitatory effects. The metabolic effects of LOLA reflected a mix of these two different processes and were concentration-dependent. We also investigated the effect of an intraperitoneal bolus injection of L-ornithine, L-aspartate or LOLA on levels of metabolites in kidney, liver and brain cortex and brain stem in mice (C57Bl6J) 1 h later. No significant changes in metabolite levels were seen following the bolus injection of L-aspartate, most likely due to rapid metabolism of aspartate before reaching the target tissue. Brain cortex glutamate was decreased by L-ornithine but no other brain effects were observed with any other compound. Kidney levels of aspartate were increased after injection of L-ornithine and LOLA which may be due to interference by ornithine with the kidney urea cycle. It is likely that without optimising chronic intravenous infusion, LOLA has minimal impact on healthy brain energy metabolism due to systemic clearance and the blood - brain barrier.

ß-Hydroxybutyrate in the Brain: One Molecule, Multiple Mechanisms.

ß-Hydroxybutyrate (ßOHB), a ketone body, is oxidised as a brain fuel. Although its contribution to energy metabolism in the healthy brain is minimal, it is an interesting metabolite which is not only oxidised but also has other direct and collateral effects which make it a molecule of interest for therapeutic purposes. In brain ßOHB can be produced in astrocytes from oxidation of fatty acids or catabolism of amino acids and is metabolised in the mitochondria of all brain cell types although uptake across the blood brain barrier is a metabolic control point. ßOHB possesses an intrinsic high heat of combustion, making it an efficient mitochondrial fuel, where it can alter the NAD+/NADH and Q/QH2 couples and reduce production of mitochondrial reactive oxygen species. It can directly interact as a signalling molecule influencing opening of K+ channels and regulation of Ca2+ channels. ßOHB is an inhibitor of histone deacetylases resulting in upregulation of genes involved in protection against oxidative stress and regulation of metabolism. It interacts with an inflammasome in immune cells to reduce production of inflammatory cytokines and reduce inflammation. Use of ßOHB as an efficient neurotherapeutic relies on increasing blood ßOHB levels so as to encourage entry of ßOHB to the brain. While use of ßOHB as a sole therapeutic is currently limited, with employment of a ketogenic diet a more widely used approach, recent development and testing of esterified forms of ßOHB have shown great promise, with the approach elevating plasma ßOHB while allowing consumption of normal diet. An improved understanding of the mechanisms by which ßOHB acts will allow better design of both diet and supplemental interventions.

ß-Hydroxybutyrate Boosts Mitochondrial and Neuronal Metabolism but is not Preferred Over Glucose Under Activated Conditions.

The ketone body, ß-hydroxybutyrate (ßOHB), is metabolised by the brain alongside the mandatory brain fuel glucose. To examine the extent and circumstances by which ßOHB can supplement glucose metabolism, we studied guinea pig cortical brain slices using increasing concentrations of [U-13C]D-ßOHB in conjunction with [1-13C]D-glucose under conditions of normo- and hypoglycaemia, as well as under high potassium (40 mmol/L K+) depolarization in normo- and hypoglycaemic conditions. The contribution of ßOHB to synthesis of GABA was also probed by inhibiting the synthesis of glutamine, a GABA precursor, with methionine sulfoximine (MSO). [U-13C]D-ßOHB at lower concentrations (0.25 and 1.25 mmol/L) stimulated mitochondrial metabolism, producing greater total incorporation of label into glutamate and GABA but did not have a similar effect in the cytosolic compartment where labelling of glutamine was reduced at 1.25 mmol/L [U-13C]D-ßOHB. At higher concentrations (2.5 mmol/L) [U-13C]D-ßOHB inhibited metabolism of [1-13C]D-glucose, and reduced total label incorporation and total metabolite pools. When glucose levels were reduced, ßOHB was able to partially restore the loss of glutamate and GABA caused by hypoglycaemia, but was not able to supplement levels of lactate, glutamine or alanine or to prevent the increase in aspartate. Under depolarizing conditions glucose was the preferred substrate over ßOHB, even in hypoglycaemic conditions where comparatively less ßOHB was incorporated except into aspartate isotopomers. Inhibition of glutamine synthesis with MSO had no significant effect on incorporation of label from [U-13C]D-ßOHB into GABA C2,1 indicating that the majority of this GABA was synthesized in GABAergic neurons from [U-13C]D-ßOHB rather than from Gln C4,5 imported from astrocytes.