Ketones inhibit mitochondrial production of reactive oxygen species production following glutamate excitotoxicity by increasing NADH oxidation

Abstract

Dietary protocols that increase serum levels of ketones, such as calorie restriction and the ketogenic diet, offer robust protection against a multitude of acute and chronic neurological diseases. The underlying mechanisms, however, remain unclear. Previous studies have suggested that the ketogenic diet may reduce free radical levels in the brain. Thus, one possibility is that ketones may mediate neuroprotection through antioxidant activity. In the present study, we examined the effects of the ketones β-hydroxybutyrate and acetoacetate on acutely dissociated rat neocortical neurons subjected to glutamate excitotoxicity using cellular electrophysiological and single-cell fluorescence imaging techniques. Further, we explored the effects of ketones on acutely isolated mitochondria exposed to high levels of calcium. A combination of β-hydroxybutyrate and acetoacetate (1 mM each) decreased neuronal death and prevented changes in neuronal membrane properties induced by 10 μM glutamate. Ketones also significantly decreased mitochondrial production of reactive oxygen species and the associated excitotoxic changes by increasing NADH oxidation in the mitochondrial respiratory chain, but did not affect levels of the endogenous antioxidant glutathione. In conclusion, we demonstrate that ketones reduce glutamate-induced free radical formation by increasing the NAD + /NADH ratio and enhancing mitochondrial respiration in neocortical neurons. This mechanism may, in part, contribute to the neuroprotective activity of ketones by restoring normal bioenergetic function in the face of oxidative stress. Keywordsglutamate; neurotoxicity; diet; mitochondria; oxidation; stress Address correspondence to: Jong M. Rho, MD., Neurology Research, NRC 4 th Fl., Barrow Neurological Institute and St. Joseph's Hospital & Medical Center, 350 W. Thomas Road, Phoenix, AZ 85013, Email: jong.rho@chw.edu. Section Editor: Molecular Neuroscience W. Sieghart, Brain Research Institute, University of Vienna, Division of Biochemistry and Molecular Biology, Spitalgasse 4, A-1090 Vienna, Austria Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Calorie restriction can decrease the risk of neurodegenerative disease and protect the brain against acute insults such as stroke (Mattson et al, 2002). Similarly, the ketogenic diet, a highfat, low-carbohydrate diet created to mimic the effects of calorie restriction, is an extremely efficacious treatment for medically intractable epilepsy Vining et al, 1998). Several metabolic changes occur during calorie restriction and the ketogenic diet, notably an increase in seru...

Protection of Hypoglycemia-Induced Neuronal Death by β-Hydroxybutyrate Involves the Preservation of Energy Levels and Decreased Production of Reactive Oxygen Species

“…6,10 An antioxidant action of KB can result from the increase in NADH oxidation and/or the improvement of mitochondrial function. 10,16 In addition, in a previous study we reported in a free cell system that both isomers of BHB can scavenge the hydroxyl radical with an IC50 of 2 to 3 mmol/L. 10 We also showed that both isomers reduce the number of Et-positive hippocampal cells during glycolysis inhibition, suggesting that they retain their capacity to scavenge ROS in a cellular system.…”

“…[6][7][8][33][34][35][36] The improvement of mitochondrial function and ATP production has been suggested by several studies as the main mechanism involved in the neuroprotective action of KB. 11,16,17,33 We also tested the protective effect of pyruvate and lactate, which can also be used by brain as alternative substrates to glucose through its metabolism by the tricarboxylic acid cycle. In agreement with previous studies, [37][38][39] we observed a reduction of neuronal death in the in vitro model by both substrates, while the effect of pyruvate was similar to that of D-BHB, lactate was less effective.…”

Molecular mechanisms of excitotoxicity and their relevance to pathogenesis of neurodegenerative diseases

“…Intracellular ROS are capable of inducing damage and, in severe cases, cell death through mitochondrial alterations leading to the release of cytochrome c [42,43] through activation of the JNK pathway [44] or by activation of nuclear factor-κB (NF-κB) transcription factors [45] . The ability to control ROS is thus critical in neurodegenerative diseases, because neuronal damage occurs when the ''oxidant-antioxidant'' balance is disturbed in favor of excess oxidative stress [46] . A recent study suggests that a ROS-scavenger effectively protected human neuroglioma against both necrotic and apoptotic cell death induced by hydrogen peroxide [47] .…”

The Neuropharmacology of the Ketogenic Diet

“…In neocortical neuron slices, the ketone bodies β-hydroxybutyrate and acetoacetate prevent cell injury induced by the oxidative stressors hydrogen peroxide and the thiol oxidant diamide [13]. These ketone bodies also inhibit glutamate-induced mitochondrial reactive oxygen species generation [14]. Oxidative stressors may contribute to the development of epilepsy (reviewed in [15]).…”