Blood-oxygenation level dependent (BOLD) image contrast in magnetic resonance imaging (MRI) has b... more Blood-oxygenation level dependent (BOLD) image contrast in magnetic resonance imaging (MRI) has been widely used in the field of functional imaging to interpret changes in focal brain activity in response to stimuli. The BOLD image contrast relies on activation-induced changes in the magnetic properties of blood (Ogawa et al, 1990; 1993a; 1993b; Shulman et al., 1993; Kennan et al., 1994). The presence of paramagnetic deoxyhemoglobin in the microvasculature creates a magnetic susceptibility difference between the vessels and the surrounding tissue, thus producing a microscopic magnetic field gradient. The microscopic field gradients affect the value of R2* (i.e., apparent transverse relaxation rate of tissue water) which can be mapped by a gradient-echo MRI sequence. The equilibrium between deoxy- and oxyhemoglobin can be shifted by altering the blood oxygenation, and since deoxy- and oxyhemoglobin are para- and diamagnetic, respectively, BOLD image contrast can be created, whereby hemoglobin acts as an endogenous MRI contrast agent. A BOLD functional MRI sequence measures the changes in R2* upon activation. The quantitative change in R2* is determined by parameters which influence the microscopic field gradient such as the geometry of vessels, static magnetic field strength, and the concentration of deoxyhemoglobin within the vessels (Ogawa et al., 1993a; 1993b; Kennan et al., 1994). The concentration of deoxyhemoglobin or the local blood oxygenation fraction (Y) in the microvasculature is determined by the regional values of cerebral metabolic rates of oxygen consumption (CMRO2), cerebral blood flow (CBF), and volume (CBV) (Ogawa et al., 1993a; 1993b; Kennan et al., 1994). While geometry and morphology of microvessels are important for the basal BOLD image contrast at a particular magnetic field strength, it is only the change in concentration of deoxyhemoglobin, due to a short-lived and/or transient physiological perturbation, that is important for being able to quantitate the BOLD signal change for functional MRI (see Appendix).
Purpose:Deuterium Metabolic Imaging (DMI) combined with [6,6’-2H2]-glucose has the potential to d... more Purpose:Deuterium Metabolic Imaging (DMI) combined with [6,6’-2H2]-glucose has the potential to detect glycogen synthesis in the liver. However, the similar chemical shifts of [6,6’-2H2]-glucose and [6,6’-2H2]-glycogen in the 2H NMR spectrum make unambiguous detection and separation difficult in vivo, in contrast to comparable approaches using 13C MRS. Here we investigate the NMR visibility of 2H-labeled glycogen to better understand its potential contribution to the observed signal in liver following administration of [6,6’-2H2]-glucose.Methods:Mice were provided drinking water containing 2H-labeled glucose. High resolution NMR analyses was performed of isolated liver glycogen in solution, before and after the addition of the glucose releasing enzyme amyloglucosidase.Results:2H-labeled glycogen was barely detectable in solution using 2H NMR, due to the very short T2 (< 2 ms) of 2H-labeled glycogen, giving a spectral line width that is more than five times as broad as that of 13C-labeled glycogen (T2 ~ 10 ms).Conclusion:2H-labeled glycogen is not detectable with 2H MRS(I) under in vivo conditions, leaving 13C MRS as the preferred technique for in vivo detection of glycogen.
A selective 'H NMR spin-echo editing method was used to detect the CCH, of GABA in rat brain in v... more A selective 'H NMR spin-echo editing method was used to detect the CCH, of GABA in rat brain in vivo before and after intravenous administration of the highly selective GABA transaminase inhibitor, gabaculine (3-amino-2,3-dihydrobenzoic acid-HCI; 100 mglkg, intravenously). The effects of the inhibitor on high energy phosphates and pH, were determined by 31P NMR. GABA levels increased approximately linearly ( r = 0.81 to 0.94; P< 0.0005) from 1.9 -+ 0.4 pmollg (pre-gabaculine; mean -+ SD) to between 6 and 8 pmollg after 4 hr at rates of accumulation of 1.1 to 2.9 pmol/hr/g. 'H NMR spectroscopic measurements of cerebral GABA and its rate of turnover of- fers a new approach in the study of GABA-mediated processes in vivo.
Pathophysiologic mechanisms underlying temporal lobe epilepsy (TLE) are still poorly understood. ... more Pathophysiologic mechanisms underlying temporal lobe epilepsy (TLE) are still poorly understood. One major hypothesis links alterations in energy metabolism to glutamate excitotoxicity associated with seizures in TLE. The purpose of this study was to determine whether changes in the activities of enzymes critical in energy and neurotransmitter metabolism contributed to the alterations in metabolic status leading to the excitotoxic effects of glutamate. Methods: Activities of four key enzymes involved in energy metabolism and glutamate cycling in the brain [aspartate aminotransferase (AAT), citrate synthase (CS), glutamate dehydrogenase (GDH), and lactate dehydrogenase (LDH)] were measured in anterolateral temporal neocortical and hippocampal tissues obtained from three different groups of medically intractable epilepsy patients having either mesial, paradoxical, or mass lesion-associated temporal lobe epilepsy (MTLE, PTLE, Ma-TLE), respectively. Results: We found that GDH activity was significantly decreased in the temporal cortex mainly in the MTLE group. A similar trend was recognized in the hippocampus of the MTLE. In all three patient groups, GDH activity was considerably lower, and AAT and LDH activities were higher in cortex of MTLE as compared with the corresponding activities in hippocampus (p < 0.05). In the MTLE cortex and hippocampus, GDH activities were negatively correlated with the duration since the first intractable seizure. Conclusions: Our results support the hypothesis suggesting major alteration in GDH activity mainly in the MTLE group. It is proposed that significant alterations in the enzyme activities may be contributing to decreased metabolism of glutamate, leading to its accumulation.
Glutamine synthetase (GS, E.C. 6.3.1.2) is a ubiquitous and highly compartmentalized enzyme that ... more Glutamine synthetase (GS, E.C. 6.3.1.2) is a ubiquitous and highly compartmentalized enzyme that is critically involved in several metabolic pathways in the brain, including the glutamineglutamate-GABA cycle and detoxification of ammonia. GS is normally localized to the cytoplasm of most astrocytes, with elevated concentrations of the enzyme being present in perivascular endfeet and in processes close to excitatory synapses. Interestingly, an increasing number of studies have indicated that the expression, distribution, or activity of brain GS is altered in several brain disorders, including Alzheimer's disease, schizophrenia, depression, suicidality, and mesial temporal lobe epilepsy (MTLE). Although the metabolic and functional sequelae of brain GS perturbations are not fully understood, it is likely that a deficiency in brain GS will have a significant biological impact due to the critical metabolic role of the enzyme. Furthermore, it is possible that restoration of GS in astrocytes lacking the enzyme could constitute a novel and highly specific therapy for these disorders. The goals of this review are to summarize key features of mammalian GS under normal conditions, and discuss the consequences of GS deficiency in brain disorders, specifically MTLE.
Increasing hypoxia tolerance in mammalian cells is potentially of major importance, but it has no... more Increasing hypoxia tolerance in mammalian cells is potentially of major importance, but it has not been feasible thus far. The disaccharide trehalose, which accumulates dramatically during heat shock, enhances thermotolerance and reduces aggregation of denatured proteins. Previous studies from our laboratory showed that over-expression of Drosophila trehalose-phosphate synthase (dtps1) increases the trehalose level and anoxia tolerance in flies. To determine whether trehalose can protect against anoxic injury in mammalian cells, we transfected the dtps1 gene into human HEK-293 cells using the recombinant plasmid pcDNA3.1(؊)-dtps1 and obtained more than 20 stable cell strains. Glucose starvation in culture showed that HEK-293 cells transfected with pcDNA3.1(؊)-dtps1 (HEK-dtps1) do not metabolize intracellular trehalose, and, interestingly, these cells accumulated intracellular trehalose during hypoxic exposure. In contrast to HEK-293 cells transfected with pcDNA3.1(؊) (HEK-v), cells with trehalose were more resistant to low oxygen stress (1% O 2 ). To elucidate how trehalose protects cells from anoxic injury, we assayed protein solubility and the amount of ubiquitinated proteins. There was three times more insoluble protein in HEK-v than in HEK-dtps1 after 3 days of exposure to low O 2 . The amount of Na ؉ -K ؉ ATPase present in the insoluble proteins dramatically increased in HEK-v cells after 2 and 3 days of exposure, whereas there was no significant change in HEK-dtps1 cells. Ubiquitinated proteins increased dramatically in HEK-v cells after 2 and 3 days of exposure but not in HEK-dtps1 cells over the same period. Our results indicate that increased trehalose in mammalian cells following transfection by the Drosophila tps1 gene protects cells from hypoxic injury. The mechanism of this protection is likely related to a decrease in protein denaturation, through protein-trehalose interactions, resulting in enhanced cellular recovery from hypoxic stress.
Using 1H spectroscopy, we measured occipital lobe GABA levels serially in 18 patients enrolled in... more Using 1H spectroscopy, we measured occipital lobe GABA levels serially in 18 patients enrolled in an ongoing open label trial of vigabatrin. Brain GABA levels were elevated twofold in patients taking vigabatrin (3 to 4 g/d) compared with nonepileptic subjects. Serial measurements suggested that brain GABA rose in proportion to vigabatrin dose up to 3 g/d. Doubling the dose from 3 to 6 g/d failed to increase brain GABA further. Serial measurements on three patients taking 6 g/d showed a gradual decrease in brain GABA in two patients over 1 to 2 years of treatment. These observations suggest that GABA synthesis may decrease at high GABA levels.
Vigabatrin is a novel antiepileptic drug designed to control seizures by raising brain y-aminobut... more Vigabatrin is a novel antiepileptic drug designed to control seizures by raising brain y-aminobutyric acid (GABA) concentrations . Seizure control is not improved significantly when the daily dose is increased beyond 50 mg/kg . Serial, in vivo measurements of GABA levels in human occipital lobe were made using 'H NMR spectroscopy before and after the start of vigabatrin treatment . We used a 2.1-T magnetic resonance imagerspectrometer and an 8-cm surface coil to examine serially a 14-cm 3 volume in the occipital lobe of 26 patients with complex partial seizures . Brain GABA content increased following the start of vigabatrin treatment up to a daily dose of 60 mg/kg. Additional increases in dose failed to increase brain GABA content further . GABA synthesis may decrease with sustained elevations of human brain GABA levels . Starting vigabatrin treatment reduced seizure frequency by >50%, from six to seven per month to three. Improved seizure control was not associated with further increases of vigabatrin dose. Increased brain GABA concentration was associated with improved seizure control . Starting vigabatrin treatment improved seizure control twofold when GABA levels increased above 1 .8 mmol/kg . Further increases in brain GABA content above 2 .5 mmol/kg provided less protection. Measuring occipital lobe GABA concentrations may predict improved seizure control when using antiepileptic drugs designed to increase brain GABA levels .
Journal of Cerebral Blood Flow and Metabolism, May 1, 1992
The concentration of intracerebral glucose as a function of plasma glucose concentration was meas... more The concentration of intracerebral glucose as a function of plasma glucose concentration was measured in rats by 13e NMR spectroscopy. Measurements were made in 20-60 min periods during the infusion of [1_13C]O glucose, when intracerebral and plasma glucose levels were at steady state. Intracerebral glucose was found to vary from 0.7 to 19 ILmol g-I wet weight as the steady state plasma glucose concentration was varied from 3 to 62 mM. A symmetric Michaelis-Menten model was fit to the brain and plasma glucose data with and without an unsaturable component, yielding the transport parame ters Km, V max' and Kd• If it is assumed that all transport is saturable (Kd = 0), then Km = 13. 9 ± 2.7 mM and
Proceedings of the National Academy of Sciences of the United States of America, Mar 1, 1985
We have used {'3C}-'H NMR spectroscopy at 360.13 MHz to resolve the 13C coupled proton resonance ... more We have used {'3C}-'H NMR spectroscopy at 360.13 MHz to resolve the 13C coupled proton resonance of glutamate and lactate in the rat brain in vivo. The time re- quired for the 13C fractional enrichment of the 4-CH2 position of brain glutamate to reach isotopic steady state was deter- mined during a continuous infusion of D-[1-'3C]glucose. Under
Three mouse lines with altered growth hormone (GH) signaling were used to study GHÕs role in adip... more Three mouse lines with altered growth hormone (GH) signaling were used to study GHÕs role in adiposity. Dwarf GH receptor knockout mice (GHR)/)) and bovine GH antagonist expressing mice (GHA) had an increased percent body fat with most of the excess fat mass accumulating in the subcutaneous region. Giant bovine GH expressing mice (bGH) had a reduced percent body fat. Only GHA mice consumed significantly more food per body weight. Serum leptin levels were significantly increased in GHA mice and decreased in bGH mice but unchanged in the GHR)/) mice. Interestingly, serum adiponectin levels were significantly increased in the GHR)/) and GHA lines but decreased in bGH mice. These data suggest that suppression or absence of GH action and enhanced GH action indeed have opposite metabolic effects in terms of adiposity. Interestingly, adiponectin levels were positively correlated with previously reported insulin sensitivity of these mice, but also positively correlated with adiposity, which is contrary to findings in other mouse models. Thus, adiponectin levels were negatively correlated with GH function suggesting a role for adiponectin in GH-induced insulin resistance.
Proceedings of the National Academy of Sciences of the United States of America, Mar 18, 1997
The cerebral tricarboxylic acid (TCA) cycle rate and the rate of glutamine synthesis were measure... more The cerebral tricarboxylic acid (TCA) cycle rate and the rate of glutamine synthesis were measured in rats in vivo under normal physiological and hyperammonemic conditions using 13 C NMR spectroscopy. In the hyperammonemic animals, blood ammonia levels were raised from control values of Ϸ0.05 mM to Ϸ0.35 mM by an intravenous ammonium acetate infusion. Once a steady-state of cerebral metabolites was established, a [1-13 C]glucose infusion was initiated, and 13 C NMR spectra acquired continuously on a 7tesla spectrometer to monitor 13 C labeling of cerebral metabolites. The time courses of glutamate and glutamine C-4 labeling were fitted to a mathematical model to yield TCA cycle rate (V TCA ) and the f lux from glutamate to glutamine through the glutamine synthetase pathway (V gln ). Under hyperammonemia the value of V TCA was 0.57 ؎ 0.16 mol͞min per g (mean ؎ SD, n ؍ 6) and was not significantly different (unpaired t test; P > 0.10) from that measured in the control animals (0.46 ؎ 0.12 mol͞min per g, n ؍ 5). Therefore, the TCA cycle rate was not significantly altered by hyperammonemia. The measured rate of glutamine synthesis under hyperammonemia was 0.43 ؎ 0.14 mol͞min per g (mean ؎ SD, n ؍ 6), which was significantly higher (unpaired t test; P < 0.01) than that measured in the control group (0.21 ؎ 0.04 mol͞ min per g, n ؍ 5). We propose that the majority of the glutamine synthetase f lux under normal physiological conditions results from neurotransmitter substrate cycling between neurons and glia. Under hyperammonemia the observed increase in glutamine synthesis is comparable to the expected increase in ammonia transport into the brain and reported measurements of glutamine eff lux under such conditions. Thus, under conditions of elevated plasma ammonia an increase in the rate of glutamine synthesis occurs as a means of ammonia detoxification, and this is superimposed on the constant rate of neurotransmitter cycling through glutamine synthetase. Animal Preparation. Two groups of male Sprague-Dawley rats were studied: a control group (weight 213 Ϯ 11 g; n ϭ 5) and a hyperammonemic group (weight 213 Ϯ 19 g; n ϭ 8). The animals The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
We determined cerebral intracellular pH in living rabbits and rats under physiologic conditions, ... more We determined cerebral intracellular pH in living rabbits and rats under physiologic conditions, using phosphorus NMR spectroscopy and new titration curves thought to be appropriate for brain. Mean values for the two species were, respectively, 7.14 +/- 0.04 (SD) and 7.13 +/- 0.03. These are toward the alkaline end of the range of values obtained by other methods, as values reported by other NMR workers also tend to be.
Proceedings of the National Academy of Sciences of the United States of America, May 1, 1981
13C NMR was used to follow the metabolism of [2-'3C]acetate and [1-_3C]acetate in aerobic suspens... more 13C NMR was used to follow the metabolism of [2-'3C]acetate and [1-_3C]acetate in aerobic suspensions of Saccharomyces cerevisiae. In the experiment with [2-'3C]acetate, the 13C label appeared first in glutamate C4 and subsequently in glutamate C2 and C3. After exhaustion of the acetate, the glutamate signals diminished and the aspartate C2 and C3 peaks increased. During a subsequent chase experiment with unlabeled acetate, the aspartate peaks decreased and the glutamate C2 and C3 peaks increased in intensity. These observations are interpreted in terms of an interplay between the glutamic-oxalacetic transaminase and Krebs cycle activity. This interpretation was confirmed by an experiment with the transaminase inhibitor 2-amino oxyacetate. During all of these experiments, we observed the formation of trehalose. The NMR gives a direct measurement of the label distribution and from that information it followed that the flows through the glyoxylate and the Krebs cycles are comparable. The intermediates citrate, succinate, fumarate, malate, phosphoenolpyruvate, 3-phosphoglycerate, and glucose 6-phosphate were identified in a 13C NMR spectrum of a perchloric acid extract taken during the metabolism of [2-'3C]acetate. Enrichment of the glutamate C5 position shows the existence of a futile cycle in which phosphoenolpyruvate, formed from oxaloacetate, returns to the Krebs cycle through pyruvate and acetyl CoA.
Regional rates of 13C incorporation from glucose to glutamate were detected in anesthetized rat b... more Regional rates of 13C incorporation from glucose to glutamate were detected in anesthetized rat brain in vivo at 7T with high temporal and spatial resolution using NMR method ICED PEPSI (in vivo carbon edited detection with proton echo planar spectroscopic imaging). Time courses of regional glutamate 13C turnover were fitted by a metabolic model to obtain regional tri-carboxylic acid (TCA) cycle flux and cerebral metabolic rate of oxygen consumption (CMRO2) in each voxel (8 microL) of rat cortex. CMRO2 maps obtained for rats under either alpha-chloralose or morphine anesthesia revealed average cortical values of 1.5 +/- 0.2 (n = 3) and 3.2 +/- 0.3 (n = 4) mumol/g/min, respectively. These values of CMRO2 are in good agreement with previous cortical measurements with coarser spatial resolution. The heterogeneity within each map, which depicted predominantly gray and white matter differences, was significantly greater under morphine (higher cortical activity) than under-alpha-chloralose (lower cortical activity) anesthesia. The regional variations in the basal awake state, which are expected to be even greater, should be considered to avoid partial-volume artifacts in functional activation studies of awake subjects.
Blood-oxygenation level dependent (BOLD) image contrast in magnetic resonance imaging (MRI) has b... more Blood-oxygenation level dependent (BOLD) image contrast in magnetic resonance imaging (MRI) has been widely used in the field of functional imaging to interpret changes in focal brain activity in response to stimuli. The BOLD image contrast relies on activation-induced changes in the magnetic properties of blood (Ogawa et al, 1990; 1993a; 1993b; Shulman et al., 1993; Kennan et al., 1994). The presence of paramagnetic deoxyhemoglobin in the microvasculature creates a magnetic susceptibility difference between the vessels and the surrounding tissue, thus producing a microscopic magnetic field gradient. The microscopic field gradients affect the value of R2* (i.e., apparent transverse relaxation rate of tissue water) which can be mapped by a gradient-echo MRI sequence. The equilibrium between deoxy- and oxyhemoglobin can be shifted by altering the blood oxygenation, and since deoxy- and oxyhemoglobin are para- and diamagnetic, respectively, BOLD image contrast can be created, whereby hemoglobin acts as an endogenous MRI contrast agent. A BOLD functional MRI sequence measures the changes in R2* upon activation. The quantitative change in R2* is determined by parameters which influence the microscopic field gradient such as the geometry of vessels, static magnetic field strength, and the concentration of deoxyhemoglobin within the vessels (Ogawa et al., 1993a; 1993b; Kennan et al., 1994). The concentration of deoxyhemoglobin or the local blood oxygenation fraction (Y) in the microvasculature is determined by the regional values of cerebral metabolic rates of oxygen consumption (CMRO2), cerebral blood flow (CBF), and volume (CBV) (Ogawa et al., 1993a; 1993b; Kennan et al., 1994). While geometry and morphology of microvessels are important for the basal BOLD image contrast at a particular magnetic field strength, it is only the change in concentration of deoxyhemoglobin, due to a short-lived and/or transient physiological perturbation, that is important for being able to quantitate the BOLD signal change for functional MRI (see Appendix).
Purpose:Deuterium Metabolic Imaging (DMI) combined with [6,6’-2H2]-glucose has the potential to d... more Purpose:Deuterium Metabolic Imaging (DMI) combined with [6,6’-2H2]-glucose has the potential to detect glycogen synthesis in the liver. However, the similar chemical shifts of [6,6’-2H2]-glucose and [6,6’-2H2]-glycogen in the 2H NMR spectrum make unambiguous detection and separation difficult in vivo, in contrast to comparable approaches using 13C MRS. Here we investigate the NMR visibility of 2H-labeled glycogen to better understand its potential contribution to the observed signal in liver following administration of [6,6’-2H2]-glucose.Methods:Mice were provided drinking water containing 2H-labeled glucose. High resolution NMR analyses was performed of isolated liver glycogen in solution, before and after the addition of the glucose releasing enzyme amyloglucosidase.Results:2H-labeled glycogen was barely detectable in solution using 2H NMR, due to the very short T2 (< 2 ms) of 2H-labeled glycogen, giving a spectral line width that is more than five times as broad as that of 13C-labeled glycogen (T2 ~ 10 ms).Conclusion:2H-labeled glycogen is not detectable with 2H MRS(I) under in vivo conditions, leaving 13C MRS as the preferred technique for in vivo detection of glycogen.
A selective 'H NMR spin-echo editing method was used to detect the CCH, of GABA in rat brain in v... more A selective 'H NMR spin-echo editing method was used to detect the CCH, of GABA in rat brain in vivo before and after intravenous administration of the highly selective GABA transaminase inhibitor, gabaculine (3-amino-2,3-dihydrobenzoic acid-HCI; 100 mglkg, intravenously). The effects of the inhibitor on high energy phosphates and pH, were determined by 31P NMR. GABA levels increased approximately linearly ( r = 0.81 to 0.94; P< 0.0005) from 1.9 -+ 0.4 pmollg (pre-gabaculine; mean -+ SD) to between 6 and 8 pmollg after 4 hr at rates of accumulation of 1.1 to 2.9 pmol/hr/g. 'H NMR spectroscopic measurements of cerebral GABA and its rate of turnover of- fers a new approach in the study of GABA-mediated processes in vivo.
Pathophysiologic mechanisms underlying temporal lobe epilepsy (TLE) are still poorly understood. ... more Pathophysiologic mechanisms underlying temporal lobe epilepsy (TLE) are still poorly understood. One major hypothesis links alterations in energy metabolism to glutamate excitotoxicity associated with seizures in TLE. The purpose of this study was to determine whether changes in the activities of enzymes critical in energy and neurotransmitter metabolism contributed to the alterations in metabolic status leading to the excitotoxic effects of glutamate. Methods: Activities of four key enzymes involved in energy metabolism and glutamate cycling in the brain [aspartate aminotransferase (AAT), citrate synthase (CS), glutamate dehydrogenase (GDH), and lactate dehydrogenase (LDH)] were measured in anterolateral temporal neocortical and hippocampal tissues obtained from three different groups of medically intractable epilepsy patients having either mesial, paradoxical, or mass lesion-associated temporal lobe epilepsy (MTLE, PTLE, Ma-TLE), respectively. Results: We found that GDH activity was significantly decreased in the temporal cortex mainly in the MTLE group. A similar trend was recognized in the hippocampus of the MTLE. In all three patient groups, GDH activity was considerably lower, and AAT and LDH activities were higher in cortex of MTLE as compared with the corresponding activities in hippocampus (p < 0.05). In the MTLE cortex and hippocampus, GDH activities were negatively correlated with the duration since the first intractable seizure. Conclusions: Our results support the hypothesis suggesting major alteration in GDH activity mainly in the MTLE group. It is proposed that significant alterations in the enzyme activities may be contributing to decreased metabolism of glutamate, leading to its accumulation.
Glutamine synthetase (GS, E.C. 6.3.1.2) is a ubiquitous and highly compartmentalized enzyme that ... more Glutamine synthetase (GS, E.C. 6.3.1.2) is a ubiquitous and highly compartmentalized enzyme that is critically involved in several metabolic pathways in the brain, including the glutamineglutamate-GABA cycle and detoxification of ammonia. GS is normally localized to the cytoplasm of most astrocytes, with elevated concentrations of the enzyme being present in perivascular endfeet and in processes close to excitatory synapses. Interestingly, an increasing number of studies have indicated that the expression, distribution, or activity of brain GS is altered in several brain disorders, including Alzheimer's disease, schizophrenia, depression, suicidality, and mesial temporal lobe epilepsy (MTLE). Although the metabolic and functional sequelae of brain GS perturbations are not fully understood, it is likely that a deficiency in brain GS will have a significant biological impact due to the critical metabolic role of the enzyme. Furthermore, it is possible that restoration of GS in astrocytes lacking the enzyme could constitute a novel and highly specific therapy for these disorders. The goals of this review are to summarize key features of mammalian GS under normal conditions, and discuss the consequences of GS deficiency in brain disorders, specifically MTLE.
Increasing hypoxia tolerance in mammalian cells is potentially of major importance, but it has no... more Increasing hypoxia tolerance in mammalian cells is potentially of major importance, but it has not been feasible thus far. The disaccharide trehalose, which accumulates dramatically during heat shock, enhances thermotolerance and reduces aggregation of denatured proteins. Previous studies from our laboratory showed that over-expression of Drosophila trehalose-phosphate synthase (dtps1) increases the trehalose level and anoxia tolerance in flies. To determine whether trehalose can protect against anoxic injury in mammalian cells, we transfected the dtps1 gene into human HEK-293 cells using the recombinant plasmid pcDNA3.1(؊)-dtps1 and obtained more than 20 stable cell strains. Glucose starvation in culture showed that HEK-293 cells transfected with pcDNA3.1(؊)-dtps1 (HEK-dtps1) do not metabolize intracellular trehalose, and, interestingly, these cells accumulated intracellular trehalose during hypoxic exposure. In contrast to HEK-293 cells transfected with pcDNA3.1(؊) (HEK-v), cells with trehalose were more resistant to low oxygen stress (1% O 2 ). To elucidate how trehalose protects cells from anoxic injury, we assayed protein solubility and the amount of ubiquitinated proteins. There was three times more insoluble protein in HEK-v than in HEK-dtps1 after 3 days of exposure to low O 2 . The amount of Na ؉ -K ؉ ATPase present in the insoluble proteins dramatically increased in HEK-v cells after 2 and 3 days of exposure, whereas there was no significant change in HEK-dtps1 cells. Ubiquitinated proteins increased dramatically in HEK-v cells after 2 and 3 days of exposure but not in HEK-dtps1 cells over the same period. Our results indicate that increased trehalose in mammalian cells following transfection by the Drosophila tps1 gene protects cells from hypoxic injury. The mechanism of this protection is likely related to a decrease in protein denaturation, through protein-trehalose interactions, resulting in enhanced cellular recovery from hypoxic stress.
Using 1H spectroscopy, we measured occipital lobe GABA levels serially in 18 patients enrolled in... more Using 1H spectroscopy, we measured occipital lobe GABA levels serially in 18 patients enrolled in an ongoing open label trial of vigabatrin. Brain GABA levels were elevated twofold in patients taking vigabatrin (3 to 4 g/d) compared with nonepileptic subjects. Serial measurements suggested that brain GABA rose in proportion to vigabatrin dose up to 3 g/d. Doubling the dose from 3 to 6 g/d failed to increase brain GABA further. Serial measurements on three patients taking 6 g/d showed a gradual decrease in brain GABA in two patients over 1 to 2 years of treatment. These observations suggest that GABA synthesis may decrease at high GABA levels.
Vigabatrin is a novel antiepileptic drug designed to control seizures by raising brain y-aminobut... more Vigabatrin is a novel antiepileptic drug designed to control seizures by raising brain y-aminobutyric acid (GABA) concentrations . Seizure control is not improved significantly when the daily dose is increased beyond 50 mg/kg . Serial, in vivo measurements of GABA levels in human occipital lobe were made using 'H NMR spectroscopy before and after the start of vigabatrin treatment . We used a 2.1-T magnetic resonance imagerspectrometer and an 8-cm surface coil to examine serially a 14-cm 3 volume in the occipital lobe of 26 patients with complex partial seizures . Brain GABA content increased following the start of vigabatrin treatment up to a daily dose of 60 mg/kg. Additional increases in dose failed to increase brain GABA content further . GABA synthesis may decrease with sustained elevations of human brain GABA levels . Starting vigabatrin treatment reduced seizure frequency by >50%, from six to seven per month to three. Improved seizure control was not associated with further increases of vigabatrin dose. Increased brain GABA concentration was associated with improved seizure control . Starting vigabatrin treatment improved seizure control twofold when GABA levels increased above 1 .8 mmol/kg . Further increases in brain GABA content above 2 .5 mmol/kg provided less protection. Measuring occipital lobe GABA concentrations may predict improved seizure control when using antiepileptic drugs designed to increase brain GABA levels .
Journal of Cerebral Blood Flow and Metabolism, May 1, 1992
The concentration of intracerebral glucose as a function of plasma glucose concentration was meas... more The concentration of intracerebral glucose as a function of plasma glucose concentration was measured in rats by 13e NMR spectroscopy. Measurements were made in 20-60 min periods during the infusion of [1_13C]O glucose, when intracerebral and plasma glucose levels were at steady state. Intracerebral glucose was found to vary from 0.7 to 19 ILmol g-I wet weight as the steady state plasma glucose concentration was varied from 3 to 62 mM. A symmetric Michaelis-Menten model was fit to the brain and plasma glucose data with and without an unsaturable component, yielding the transport parame ters Km, V max' and Kd• If it is assumed that all transport is saturable (Kd = 0), then Km = 13. 9 ± 2.7 mM and
Proceedings of the National Academy of Sciences of the United States of America, Mar 1, 1985
We have used {'3C}-'H NMR spectroscopy at 360.13 MHz to resolve the 13C coupled proton resonance ... more We have used {'3C}-'H NMR spectroscopy at 360.13 MHz to resolve the 13C coupled proton resonance of glutamate and lactate in the rat brain in vivo. The time re- quired for the 13C fractional enrichment of the 4-CH2 position of brain glutamate to reach isotopic steady state was deter- mined during a continuous infusion of D-[1-'3C]glucose. Under
Three mouse lines with altered growth hormone (GH) signaling were used to study GHÕs role in adip... more Three mouse lines with altered growth hormone (GH) signaling were used to study GHÕs role in adiposity. Dwarf GH receptor knockout mice (GHR)/)) and bovine GH antagonist expressing mice (GHA) had an increased percent body fat with most of the excess fat mass accumulating in the subcutaneous region. Giant bovine GH expressing mice (bGH) had a reduced percent body fat. Only GHA mice consumed significantly more food per body weight. Serum leptin levels were significantly increased in GHA mice and decreased in bGH mice but unchanged in the GHR)/) mice. Interestingly, serum adiponectin levels were significantly increased in the GHR)/) and GHA lines but decreased in bGH mice. These data suggest that suppression or absence of GH action and enhanced GH action indeed have opposite metabolic effects in terms of adiposity. Interestingly, adiponectin levels were positively correlated with previously reported insulin sensitivity of these mice, but also positively correlated with adiposity, which is contrary to findings in other mouse models. Thus, adiponectin levels were negatively correlated with GH function suggesting a role for adiponectin in GH-induced insulin resistance.
Proceedings of the National Academy of Sciences of the United States of America, Mar 18, 1997
The cerebral tricarboxylic acid (TCA) cycle rate and the rate of glutamine synthesis were measure... more The cerebral tricarboxylic acid (TCA) cycle rate and the rate of glutamine synthesis were measured in rats in vivo under normal physiological and hyperammonemic conditions using 13 C NMR spectroscopy. In the hyperammonemic animals, blood ammonia levels were raised from control values of Ϸ0.05 mM to Ϸ0.35 mM by an intravenous ammonium acetate infusion. Once a steady-state of cerebral metabolites was established, a [1-13 C]glucose infusion was initiated, and 13 C NMR spectra acquired continuously on a 7tesla spectrometer to monitor 13 C labeling of cerebral metabolites. The time courses of glutamate and glutamine C-4 labeling were fitted to a mathematical model to yield TCA cycle rate (V TCA ) and the f lux from glutamate to glutamine through the glutamine synthetase pathway (V gln ). Under hyperammonemia the value of V TCA was 0.57 ؎ 0.16 mol͞min per g (mean ؎ SD, n ؍ 6) and was not significantly different (unpaired t test; P > 0.10) from that measured in the control animals (0.46 ؎ 0.12 mol͞min per g, n ؍ 5). Therefore, the TCA cycle rate was not significantly altered by hyperammonemia. The measured rate of glutamine synthesis under hyperammonemia was 0.43 ؎ 0.14 mol͞min per g (mean ؎ SD, n ؍ 6), which was significantly higher (unpaired t test; P < 0.01) than that measured in the control group (0.21 ؎ 0.04 mol͞ min per g, n ؍ 5). We propose that the majority of the glutamine synthetase f lux under normal physiological conditions results from neurotransmitter substrate cycling between neurons and glia. Under hyperammonemia the observed increase in glutamine synthesis is comparable to the expected increase in ammonia transport into the brain and reported measurements of glutamine eff lux under such conditions. Thus, under conditions of elevated plasma ammonia an increase in the rate of glutamine synthesis occurs as a means of ammonia detoxification, and this is superimposed on the constant rate of neurotransmitter cycling through glutamine synthetase. Animal Preparation. Two groups of male Sprague-Dawley rats were studied: a control group (weight 213 Ϯ 11 g; n ϭ 5) and a hyperammonemic group (weight 213 Ϯ 19 g; n ϭ 8). The animals The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
We determined cerebral intracellular pH in living rabbits and rats under physiologic conditions, ... more We determined cerebral intracellular pH in living rabbits and rats under physiologic conditions, using phosphorus NMR spectroscopy and new titration curves thought to be appropriate for brain. Mean values for the two species were, respectively, 7.14 +/- 0.04 (SD) and 7.13 +/- 0.03. These are toward the alkaline end of the range of values obtained by other methods, as values reported by other NMR workers also tend to be.
Proceedings of the National Academy of Sciences of the United States of America, May 1, 1981
13C NMR was used to follow the metabolism of [2-'3C]acetate and [1-_3C]acetate in aerobic suspens... more 13C NMR was used to follow the metabolism of [2-'3C]acetate and [1-_3C]acetate in aerobic suspensions of Saccharomyces cerevisiae. In the experiment with [2-'3C]acetate, the 13C label appeared first in glutamate C4 and subsequently in glutamate C2 and C3. After exhaustion of the acetate, the glutamate signals diminished and the aspartate C2 and C3 peaks increased. During a subsequent chase experiment with unlabeled acetate, the aspartate peaks decreased and the glutamate C2 and C3 peaks increased in intensity. These observations are interpreted in terms of an interplay between the glutamic-oxalacetic transaminase and Krebs cycle activity. This interpretation was confirmed by an experiment with the transaminase inhibitor 2-amino oxyacetate. During all of these experiments, we observed the formation of trehalose. The NMR gives a direct measurement of the label distribution and from that information it followed that the flows through the glyoxylate and the Krebs cycles are comparable. The intermediates citrate, succinate, fumarate, malate, phosphoenolpyruvate, 3-phosphoglycerate, and glucose 6-phosphate were identified in a 13C NMR spectrum of a perchloric acid extract taken during the metabolism of [2-'3C]acetate. Enrichment of the glutamate C5 position shows the existence of a futile cycle in which phosphoenolpyruvate, formed from oxaloacetate, returns to the Krebs cycle through pyruvate and acetyl CoA.
Regional rates of 13C incorporation from glucose to glutamate were detected in anesthetized rat b... more Regional rates of 13C incorporation from glucose to glutamate were detected in anesthetized rat brain in vivo at 7T with high temporal and spatial resolution using NMR method ICED PEPSI (in vivo carbon edited detection with proton echo planar spectroscopic imaging). Time courses of regional glutamate 13C turnover were fitted by a metabolic model to obtain regional tri-carboxylic acid (TCA) cycle flux and cerebral metabolic rate of oxygen consumption (CMRO2) in each voxel (8 microL) of rat cortex. CMRO2 maps obtained for rats under either alpha-chloralose or morphine anesthesia revealed average cortical values of 1.5 +/- 0.2 (n = 3) and 3.2 +/- 0.3 (n = 4) mumol/g/min, respectively. These values of CMRO2 are in good agreement with previous cortical measurements with coarser spatial resolution. The heterogeneity within each map, which depicted predominantly gray and white matter differences, was significantly greater under morphine (higher cortical activity) than under-alpha-chloralose (lower cortical activity) anesthesia. The regional variations in the basal awake state, which are expected to be even greater, should be considered to avoid partial-volume artifacts in functional activation studies of awake subjects.
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