Evaluation of brain mitochondrial glutamate and alpha-ketoglutarate transport under physiologic conditions

Some models of brain energy metabolism used to interpret in vivo (13)C nuclear magnetic resonance spectroscopic data assume that intramitochondrial alpha-ketoglutarate is in rapid isotopic equilibrium with total brain glutamate, most of which is cytosolic. If so, the kinetics of changes in (13)C-glutamate can be used to predict citric acid cycle flux. For this to be a valid assumption, the brain mitochondrial transporters of glutamate and alpha-ketoglutarate must operate under physiologic conditions at rates much faster than that of the citric acid cycle. To test the assumption, we incubated brain mitochondria under physiologic conditions, metabolizing both pyruvate and glutamate and measured rates of glutamate, aspartate, and alpha-ketoglutarate transport. Under the conditions employed (66% of maximal O(2) consumption), the rate of synthesis of intramitochondrial alpha-ketoglutarate was 142 nmol/min.mg and the combined initial rate of alpha-ketoglutarate plus glutamate efflux from the mitochondria was 95 nmol/min.mg. It thus seems that much of the alpha-ketoglutarate synthesized within the mitochondria proceeds around the citric acid cycle without equilibrating with cytosolic glutamate. Unless the two pools are in such rapid exchange that they maintain the same percent (13)C enrichment at all points, (13)C enrichment of glutamate alone cannot be used to determine tricarboxylic acid cycle flux. The alpha-ketoglutarate pool is far smaller than the glutamate pool and will therefore approach steady state faster than will glutamate at the metabolite transport rates measured

Mechanism of age-dependent susceptibility and novel treatment strategy in glutaric acidemia type I

Glutaric acidemia type I (GA-I) is an inherited disorder of lysine and tryptophan metabolism presenting with striatal lesions anatomically and symptomatically similar to Huntington disease. Affected children commonly suffer acute brain injury in the context of a catabolic state associated with nonspecific illness. The mechanisms underlying injury and age-dependent susceptibility have been unknown, and lack of a diagnostic marker heralding brain injury has impeded intervention efforts. Using a mouse model of GA-I, we show that pathologic events began in the neuronal compartment while enhanced lysine accumulation in the immature brain allowed increased glutaric acid production resulting in age-dependent injury. Glutamate and GABA depletion correlated with brain glutaric acid accumulation and could be monitored in vivo by proton nuclear magnetic resonance (1H NMR) spectroscopy as a diagnostic marker. Blocking brain lysine uptake reduced glutaric acid levels and brain injury. These findings provide what we believe are new monitoring and treatment strategies that may translate for use in human GA-I

Neuroglial metabolism in the awake rat brain: CO2 fixation increases with brain activity

Glial cells are thought to supply energy for neurotransmission by increasing nonoxidative glycolysis; however, oxidative metabolism in glia may also contribute to increased brain activity. To study glial contribution to cerebral energy metabolism in the unanesthetized state, we measured neuronal and glial metabolic fluxes in the awake rat brain by using a double isotopic-labeling technique and a two-compartment mathematical model of neurotransmitter metabolism. Rats (n = 23) were infused simultaneously with 14C-bicarbonate and [1-13C]glucose for up to 1 hr. The 14C and 13C labeling of glutamate, glutamine, and aspartate was measured at five time points in tissue extracts using scintillation counting and 13C nuclear magnetic resonance of the chromatographically separated amino acids. The isotopic 13C enrichment of glutamate and glutamine was different, suggesting significant rates of glial metabolism compared with the glutamate-glutamine cycle. Modeling the 13C-labeling time courses alone and with 14C confirmed significant glial TCA cycle activity (V(PDH)((g)), approximately 0.5 micromol x gm(-1) x min(-1)) relative to the glutamate-glutamine cycle (V(NT)) (approximately 0.5-0.6 micromol x gm(-1) x min(-1)). The glial TCA cycle rate was approximately 30% of total TCA cycle activity. A high pyruvate carboxylase rate (V(PC), approximately 0.14-0.18 micromol x gm(-1) x min(-1)) contributed to the glial TCA cycle flux. This anaplerotic rate in the awake rat brain was severalfold higher than under deep pentobarbital anesthesia, measured previously in our laboratory using the same 13C-labeling technique. We postulate that the high rate of anaplerosis in awake brain is linked to brain activity by maintaining glial glutamine concentrations during increased neurotransmission

Whole genome assessment of the retinal response to diabetes reveals a progressive neurovascular inflammatory response

<p>Abstract</p> <p>Background</p> <p>Despite advances in the understanding of diabetic retinopathy, the nature and time course of molecular changes in the retina with diabetes are incompletely described. This study characterized the functional and molecular phenotype of the retina with increasing durations of diabetes.</p> <p>Results</p> <p>Using the streptozotocin-induced rat model of diabetes, levels of retinal permeability, caspase activity, and gene expression were examined after 1 and 3 months of diabetes. Gene expression changes were identified by whole genome microarray and confirmed by qPCR in the same set of animals as used in the microarray analyses and subsequently validated in independent sets of animals. Increased levels of vascular permeability and caspase-3 activity were observed at 3 months of diabetes, but not 1 month. Significantly more and larger magnitude gene expression changes were observed after 3 months than after 1 month of diabetes. Quantitative PCR validation of selected genes related to inflammation, microvasculature and neuronal function confirmed gene expression changes in multiple independent sets of animals.</p> <p>Conclusion</p> <p>These changes in permeability, apoptosis, and gene expression provide further evidence of progressive retinal malfunction with increasing duration of diabetes. The specific gene expression changes confirmed in multiple sets of animals indicate that pro-inflammatory, anti-vascular barrier, and neurodegenerative changes occur in tandem with functional increases in apoptosis and vascular permeability. These responses are shared with the clinically documented inflammatory response in diabetic retinopathy suggesting that this model may be used to test anti-inflammatory therapeutics.</p