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...

Ketone bodies (KB) have been shown to prevent neurodegeneration in models of Parkinson's and Alzheimer's diseases, but the mechanisms underlying these effects remain unclear. One possibility is that KB may exert antioxidant activity. In the current study, we explored the effects of KB on rat neocortical neurons exposed to hydrogen peroxide (H 2 O 2 ) or diamide -a thiol oxidant and activator of mitochondrial permeability transition (mPT). We found that: (i) KB completely blocked large inward currents induced by either H 2 O 2 or diamide; (ii) KB significantly decreased the number of propidium iodide-labeled cells in neocortical slices after exposure to H 2 O 2 or diamide; (iii) KB significantly decreased reactive oxygen species (ROS) levels in dissociated neurons and in isolated neocortical mitochondria; (iv) the electrophysiological effects of KB in neurons exposed to H 2 O 2 or diamide were mimicked by bongkrekic acid and cyclosporin A, known inhibitors of mPT, as well as by catalase and DLdithiothreitol, known antioxidants; (v) diamide alone did not significantly alter basal ROS levels in neurons, supporting previous studies indicating that diamide-induced neuronal injury may be mediated by mPT opening; and (vi) KB significantly increased the threshold for calcium-induced mPT in isolated mitochondria. Taken together, our data suggest that KB may prevent mPT and oxidative injury in neocortical neurons, most likely by decreasing mitochondrial ROS production.

To determine the neuroprotective effect of fasting after traumatic brain injury (TBI) and to elucidate the potential underlying mechanisms, we used a controlled cortical impact (CCI) injury model to induce either a moderate or a severe injury to adult male Sprague Dawley rats. Tissue-sparing assessments were used to determine the level of neuroprotection of fasting, hypoglycemia (insulin 10 U), or ketone (1.66 mmoles/kg/day or 0.83 mmoles/kg/day; D-beta-hydroxtbutyrate) administration. Mitochondrial isolation and respiratory studies were utilized to determine the functionality of mitochondria at the site of injury. We also investigated biomarkers of oxidative stress, such as lipid/protein oxidation, reactive oxygen species (ROS) production, and intramitochondrial calcium load, as a secondary measure of mitochondrial homeostasis. We found that fasting animals for 24 hr, but not 48 hr, after a moderate (1.5 mm), but not severe (2.0 mm), CCI resulted in a significant increase in tissue sparing. This 24-hr fast also decreased biomarkers of oxidative stress and calcium loading and increased mitochondrial oxidative phosphorylation in mitochondria isolated from the site of injury. Insulin administration, designed to mimic the hypoglycemic effect seen during fasting did not result in significant tissue sparing after moderate CCI injury and in fact induced increased mortality at some injection time points. However, the administration of ketones resulted in increased tissue sparing after moderate injury. Fasting for 24 hr confers neuroprotection, maintains cognitive function, and improves mitochondrial function after moderate (1.5 mm) TBI. The underlying mechanism appears to involve ketosis rather than hypoglycemia.

No abstract

SUMMARYUncoupling proteins (UCP) translocate protons from the mitochondrial intermembrane space to the matrix, thereby "uncoupling" electron transport from the production of ATP. It has been shown that these proteins are highly expressed in animals maintained on the ketogenic diet (KD). Although the exact mechanism remains unclear, it is known that these proteins are activated within a protective antireactive oxygen species (ROS) mechanism by free fatty acids (FFA). In our current studies, mitochondrial samples were probed for the presence of UCP2, which is the most ubiquitously expressed UCP isoform. We found that both traumatic brain injury and fasting upregulated the expression of UCP2, with a synergistic upregulation in fasted injured animals. We then used mitochondria from fasted naive animals to screen a number of FFA for their activation of uncoupling as well as their ability to reduce ROS. We found that arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), palmitoleic acid, myristic acid, and butyric acid increased mitochondrial uncoupling when added after oligomycin. These FFA, along with oleic acid, also reduced ROS in mitochondria incubated with oligomycin. In order to correlate our data to KD and fasting, both of which have been shown to be neuroprotective after neurologic insult, we determined the serum levels of FFA in KD and fasted animals using gas chromatography/mass spectroscopy. We also determined brain and cerebrospinal fluid (CSF) FFA levels from fasted animals.