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The mechanistic link of ketosis to neuroprotection under certain pathological conditions continues to be explored. We investigated whether chronic ketosis induced by ketogenic diet results in the partitioning of ketone bodies toward oxidative metabolism in brain. We hypothesized that diet-induced ketosis results in increased shunting of ketone bodies toward citric acid cycle and amino acids with decreased carbon shunting from glucose. Rats were fed standard (STD) or ketogenic (KG) diets for 3.5 weeks and then infused with [U-(13) C]glucose or [U-(13) C]acetoacetate tracers. Concentrations and (13) C-labeling pattern of citric acid cycle intermediates and amino acids were analyzed from brain homogenates using stable isotopomer mass spectrometry analysis. The contribution of [U-(13) C]glucose to acetyl-CoA and amino acids decreased by ~ 30% in the KG group versus STD, whereas [U-(13) C]acetoacetate contributions were more than two-fold higher. The concentration of GABA remained constant across groups; however, the (13) C labeling of GABA was markedly increased in the KG group infused with [U-(13) C]acetoacetate compared to STD. This study reveals that there is a significant contribution of ketone bodies to oxidative metabolism and GABA in diet-induced ketosis. We propose that this represents a fundamental mechanism of neuroprotection under pathological conditions., (© 2014 International Society for Neurochemistry.)

The brain is dependent on glucose as a primary energy substrate, but is capable of utilizing ketones such as β-hydroxybutyrate and acetoacetate, as occurs with fasting, starvation, or chronic feeding of a ketogenic diet. The relationship between changes in cerebral metabolic rates of glucose (CMRglc) and degree or duration of ketosis remains uncertain. To investigate if CMRglc decreases with chronic ketosis, 2-[(18)F]fluoro-2-deoxy-D-glucose in combination with positron emission tomography, was applied in anesthetized young adult rats fed 3 weeks of either standard or ketogenic diets. Cerebral metabolic rates of glucose (μmol/min per 100 g) was determined in the cerebral cortex and cerebellum using Gjedde-Patlak analysis. The average CMRglc significantly decreased in the cerebral cortex (23.0±4.9 versus 32.9±4.7) and cerebellum (29.3±8.6 versus 41.2±6.4) with increased plasma ketone bodies in the ketotic rats compared with standard diet group. The reduction of CMRglc in both brain regions correlates linearly by ∼9% for each 1 mmol/L increase of total plasma ketone bodies (0.3 to 6.3 mmol/L). Together with our meta-analysis, these data revealed that the degree and duration of ketosis has a major role in determining the corresponding change in CMRglc with ketosis.

Unlabelled: Ketone bodies are an alternative energy substrate to glucose in brain. Under conditions of oxidative stress, we hypothesize that ketosis stabilizes glucose metabolism by partitioning glucose away from oxidative metabolism towards ketone body oxidation. In this study we assessed oxidative metabolism in ketotic rat brain using stable isotope mass spectrometry analysis. The contribution of glucose and ketone bodies to oxidative metabolism was studied in cortical brain homogenates isolated from anesthetized ketotic rats. To induce chronic ketosis, rats were fed either a ketogenic (high-fat, carbohydrate restricted) or standard rodent chow for 3 weeks and then infused intravenously with tracers of [U-(13)C] glucose or [U-(13)C] acetoacetate for 60 min. The measured percent contribution of glucose or ketone bodies to oxidative metabolism was analyzed by measuring the (13)C-label incorporation into acetyl-CoA. Using mass spectrometry (gas-chromatography; GC-MS, and liquid-chromatography; LCMS) and isotopomer analysis, the fractional amount of substrate oxidation was measured as the M + 2 enrichment (%) of acetyl-CoA relative to the achieved enrichment of the infused precursors, [U-(13)C]glucose or [U-(13)C] acetoacetate., Results: the percent contribution of glucose oxidation in cortical brain in rats fed the ketogenic diet was 71.2 ± 16.8 (mean% ± SD) compared to the standard chow, 89.0 ± 14.6. Acetoacetate oxidation was significantly higher with ketosis compared to standard chow, 41.7 ± 9.4 vs. 21.9 ± 10.6. These data confer the high oxidative capacity for glucose irrespective of ketotic or non-ketotic states. With ketosis induced by 3 weeks of diet, cortical brain utilizes twice as much acetoacetate compared to non-ketosis.

Diabetes is associated with extensive microvascular pathology and decreased expression of the glucose transporter (GLUT-1) in retina, but not brain. To explore the basis of these differences, the authors simultaneously measured glucose influx (micromol x g(-1) x min(-1)) and blood flow (mL x g(-1) x min(-1)) in retina and brain cortex of nondiabetic control rats (normoglycemic and acute-hyperglycemic) and in rats with streptozotocin-induced diabetes (with or without aminoguanidine (AMG) treatment) using a single-pass, dual-label indicator method. In addition, tissue glucose and adenosine triphosphate (nmol/mg dry weight) levels were measured. Glucose influx in retina exceeded that of cortex by about threefold for both the nondiabetic and diabetic groups. In contrast, blood flow in retina was significantly lower than in cortex by about threefold for each group. Retinal and cortical glucose influx in the diabetic rats was lower than in the nondiabetic acute-hyperglycemic group, but not in the normoglycemic group. Blood flow in these tissues remained relatively unchanged with glycemic conditions. The glucose levels in the diabetic retina (aminoguanidine untreated and aminoguanidine treated) were fourfold to sixfold greater than the nondiabetic retina. The cortical glucose levels remained unchanged in all groups. These data suggest that the accumulation of glucose in the diabetic retina cannot be explained by increased endothelial-glucose uptake or increased vascular membrane permeability.

The contribution of gluconeogenesis to glucose production in vivo can be measured by enriching body water with 0.5% 2H2O and measuring the glucose labeling ratio C6/C2 (Landau et al., J. Clin. Invest. 95, 172-178, 1995). We present further refinements of the measurements of the 2H enrichments on C6 and C2 of glucose. The transfer of 2H from C6 of glucose to hexamethylenetetramine (HMT) and extraction in preparation for gas chromatography-mass spectrometry can be done in a single test tube, without distillation of the intermediate formaldehyde. In addition, extraction of small amounts of HMT is greatly improved by making a HMT-iodine adduct. For C2, glucose is reduced to sorbitol, and 2H on C2 is transferred enzymatically to [U-13C3]pyruvate, forming [U-13C3,2-2H]lactate. The latter is assayed by negative chemical ionization gas chromatography-mass spectrometry of the pentafluorobenzyl derivative. The natural enrichment of the [U-13C3]lactyl ion is only 0.4%, allowing measurements of 2H enrichment down to 0.1%. These techniques were used in dogs infused with 2H2O and in isolated rat livers perfused with buffer containing 1 to 5% 2H2O. Our data reveal a difference in the rate of labeling of C6 and C2 of glucose in vivo. Lastly, in cows infused with [6,6-2H2]glucose, we show that the turnover of glucose can be economically measured by assaying low tracer enrichment (down to 0.1%) via hexamethylenetetramine.