Increased O-GlcNAcylation rapidly decreases GABAAR currents in hippocampus but depresses neuronal output.

O-GlcNAcylation, a post-translational modification involving O-linkage of ß-N-acetylglucosamine to Ser/Thr residues on target proteins, is increasingly recognized as a critical regulator of synaptic function. Enzymes that catalyze O-GlcNAcylation are found at both presynaptic and postsynaptic sites, and O-GlcNAcylated proteins localize to synaptosomes. An acute increase in O-GlcNAcylation can affect neuronal communication by inducing long-term depression (LTD) of excitatory transmission at hippocampal CA3-CA1 synapses, as well as suppressing hyperexcitable circuits in vitro and in vivo. Despite these findings, to date, no studies have directly examined how O-GlcNAcylation modulates the efficacy of inhibitory neurotransmission. Here we show an acute increase in O-GlcNAc dampens GABAergic currents onto principal cells in rodent hippocampus likely through a postsynaptic mechanism, and has a variable effect on the excitation/inhibition balance. The overall effect of increased O-GlcNAc is reduced synaptically-driven spike probability via synaptic depression and decreased intrinsic excitability. Our results position O-GlcNAcylation as a novel regulator of the overall excitation/inhibition balance and neuronal output.

Chronic ingestion of a Western diet increases O-linked-ß-N-acetylglucosamine (O-GlcNAc) protein modification in the rat heart.

AIMS: Protein O-GlcNAcylation is both a nutrient sensing and cellular stress response that mediates signal transduction in the heart. Chronically elevated O-GlcNAc has been associated with the development of cardiac dysfunction at both the cellular and organ levels in obesity, insulin resistance and diabetes. Development of these pathologies is often attributed to diets high in saturated fat and sugar (a "Western" diet; WES) but a role for O-GlcNAc in diet-induced cardiac dysfunction has not been established. The aims of this study were to examine the effect of chronic consumption of WES on cardiac O-GlcNAcylation and investigate associations of O-GlcNAc with cardiac function and markers of cellular stress. MAIN METHODS: Young male rats received either a control diet (CON; n=9) or WES (n=8) diet for 52 weeks. KEY FINDINGS: There was no evidence of cardiac dysfunction, advanced glycation endproduct (AGE) accumulation, pathological cardiac hypertrophy, calcium handling impairment, fibrosis or endoplasmic reticulum stress in WES hearts. However, cardiac O-GlcNAc protein, particularly in the higher molecular weight range, was significantly higher in WES hearts compared to CON (P<0.05). Protein levels of the enzymes that regulate O-GlcNAc attachment were not different between groups; thus, the increased O-GlcNAcylation in WES hearts appears to be due to increased nutrient availability rather than enzymatic regulation of cellular stress. SIGNIFICANCE: These data suggest that diets high in saturated fat and sugar may contribute to the adverse effects of metabolic syndrome and diabetes by an O-GlcNAc-mediated process and that this may occur in the absence of overt cellular stress.

Myocardial phenotyping using isotopomer analysis of metabolic fluxes.

Over the past 20 years, stable isotopes combined with isotopomer analysis have proven to be a powerful approach to probe the dynamics of metabolism in various biological systems, including the heart. The aim of this paper is to demonstrate how isotopomer analysis of metabolic fluxes can provide novel insights into the myocardial phenotype. Specifically, building on our past experience using NMR spectroscopy and GC-MS as applied to investigations of cardiac energy metabolism, we highlight specific complex metabolic networks that would not be predicted by classical biochemistry or by static measurements of metabolite, protein and mRNA levels.

Evidence of separate pathways for lactate uptake and release by the perfused rat heart.

The simultaneous release and uptake of lactate by the heart has been observed both in vivo and ex vivo; however, the pathways underlying these observations have not been satisfactorily explained. Consequently, the purpose of this study was to test the hypothesis that hearts release lactate from glycolysis while simultaneously taking up exogenous lactate. Therefore, we determined the effects of fatty acids and diabetes on the regulation of lactate uptake and release. Hearts from control and 1-wk diabetic animals were perfused with 5 mM glucose, 0.5 mM [3-(13)C]lactate, and 0, 0.1, 0.32, or 1.0 mM palmitate. Parameters measured include perfusate lactate concentrations, fractional enrichment, and coronary flow rates, which enabled the simultaneous, but independent, measurements of the rates of 1) uptake of exogenous [(13)C]lactate and 2) efflux of unlabeled lactate from metabolism of glucose. Although the rates of lactate uptake and efflux were both similarly inhibited by the addition of palmitate, (i.e., the ratio of lactate uptake to efflux remained constant), the ratio of lactate uptake to efflux was significantly higher in the controls compared with the diabetic group (1.00 +/- 0.14 vs. 0.50 +/- 0.07, P < 0.002). These data, combined with heterogeneous (13)C enrichment of tissue lactate, pyruvate, and alanine, suggest that glycolytically derived lactate production and oxidation of exogenous lactate operate as functionally separate metabolic pathways. These results are consistent with the concept of an intracellular lactate shuttle.

Nuclear magnetic resonance spectroscopy and imaging in animal research.

Nuclear magnetic resonance (NMR) spectroscopy and imaging can be used to investigate, noninvasively, a wide range of biological processes in systems as diverse as protein solutions, single cells, isolated perfused organs, and tissues in vivo. It is also possible to combine different NMR techniques enabling metabolic, anatomical, and physiological information to be obtained in the same experiment. This review provides a simple overview of the basic principles of NMR and outlines both the advantages and disadvantages of NMR spectroscopy and imaging. A few examples of potential applications of NMR spectroscopy and imaging are presented, which demonstrate the range of questions that can be asked using these techniques. The potential impact of using NMR techniques in a biomedical research program on the total number of animals required for specific investigations, as well as the number of animals used in research, are discussed. The article concludes with a personal perspective on the impact of continuing improvements in NMR technology for future applications in animal research.

Upregulation of glucose metabolism during intimal lesion formation is coupled to the inhibition of vascular smooth muscle cell apoptosis. Role of GSK3beta.

The purpose of this study was to define the role of metabolic regulatory genes in the pathogenesis of vascular lesions. The glucose transporter isoform, GLUT1, was significantly increased in the neointima after balloon injury. To define the role of GLUT1 in vascular biology, we established cultured vascular smooth muscle cells (VSMCs) with constitutive upregulation of GLUT1, which led to a threefold increase in glucose uptake as well as significant increases in both nonoxidative and oxidative glucose metabolism as assessed by 13C-nuclear magnetic resonance spectroscopy. We hypothesized that the differential enhancement of glucose metabolism in the neointima contributed to formation of lesions by increasing the resistance of VSMCs to apoptosis. Indeed, upregulation of GLUT1 significantly inhibited apoptosis induced by serum withdrawal (control 20 +/- 1% vs. GLUT1 11 +/- 1%, P < 0.0005) as well as Fas-ligand (control 12 +/- 1% vs. GLUT1 6 +/- 1.0%, P < 0.0005). Provocatively, the enhanced glucose metabolism in GLUT1 overexpressing VSMC as well as neointimal tissue correlated with the inactivation of the proapoptotic kinase, glycogen synthase kinase 3beta (GSK3beta). Transient overexpression of GSK3beta was sufficient to induce apoptosis (control 7 +/- 1% vs. GSK3beta 28 +/- 2%, P < 0.0001). GSK3beta-induced apoptosis was significantly attenuated by GLUT1 overexpression (GSK3beta 29 +/- 3% vs. GLUT1 + GSK3beta 6 +/- 1%, n = 12, P < 0.001), suggesting that the antiapoptotic effect of enhanced glucose metabolism is linked to the inactivation of GSK3beta. Taken together, upregulation of glucose metabolism during intimal lesion formation promotes an antiapoptotic signaling pathway that is linked to the inactivation of GSK3beta.

Preservation of glucose metabolism in hypertrophic GLUT4-null hearts.

GLUT4-null mice lacking the insulin-sensitive glucose transporter are not diabetic but do exhibit abnormalities in glucose and lipid metabolism. The most striking morphological consequence of ablating GLUT4 is cardiac hypertrophy. GLUT4-null hearts display characteristics of hypertrophy caused by hypertension. However, GLUT4-null mice have normal blood pressure and maintain a normal cardiac contractile protein profile. Unexpectedly, although they lack the predominant glucose transporter in the heart, GLUT4-null hearts transport glucose and synthesize glycogen at normal levels, but gene expression of rate-limiting enzymes involved in fatty acid oxidation is decreased. The GLUT4-null heart represents a unique model of hypertrophy that may be used to study the consequences of altered substrate utilization in normal and pathophysiological conditions.

Preferential inhibition of lactate oxidation relative to glucose oxidation in the rat heart following diabetes.

OBJECTIVE: Alterations in myocardial metabolism occur early after the onset of diabetes suggesting that they may play a role in the development of cardiac dysfunction. Inhibition of myocardial pyruvate dehydrogenase (PDH), glucose transport and glycolysis have all been reported following diabetes. In vivo lactate is also a potential source of energy for the heart and its oxidation should not be affected by changes in glucose transport and glycolysis. Therefore, the objective of this study, was to test the hypothesis that following diabetes the inhibition of glucose oxidation would be greater than the inhibition of lactate oxidation. METHODS: Hearts from control and one-week-old diabetic rats were perfused with [1-13C]glucose (11 mmol/l) alone, [1-13C]glucose plus lactate (0.5 mmol/l) or glucose plus [3-13C]lactate (0.5 or 1.0 mmol/l) as substrates. Glucose and lactate oxidation rates were determined by combining 13C-NMR glutamate isotopomer analysis of tissue extracts with measurements of oxygen consumption. RESULTS: In diabetic hearts perfused with glucose alone, glucose oxidation was decreased compared to controls (0.31 +/- 0.08 vs. 0.71 +/- 0.11 mumoles/min/g wet weight; p < 0.05). Surprisingly, in hearts perfused with glucose plus 0.5 mmol/l lactate, there was no difference in glucose oxidation between control and diabetic groups (0.20 +/- 0.05 vs. 0.16 +/- 0.04 mumoles/min/g wet weight respectively). However, under these conditions lactate oxidation was markedly reduced in the diabetic group (0.89 +/- 0.18 vs. 0.24 +/- 0.05 mumoles/min/g wet weight; p < 0.05). At 1.0 mmol/l lactate oxidation was still significantly depressed in the diabetic group. CONCLUSION: There was a greater decrease in lactate oxidation relative to glucose oxidation in hearts from diabetic animals. These results demonstrate that diabetes leads to a specific inhibition of lactate oxidation independent of its effects on pyruvate dehydrogenase.

Impact of 1 wk of diabetes on the regulation of myocardial carbohydrate and fatty acid oxidation.

The aim of this study was to investigate the effect of increasing exogenous palmitate concentration on carbohydrate and palmitate oxidation in hearts from control and 1-wk diabetic rats. Hearts were perfused with glucose, [3-(13)C]lactate, and [U-(13)C]palmitate. Substrate oxidation rates were determined by combining (13)C-NMR glutamate isotopomer analysis of tissue extracts with measurements of oxygen consumption. Carbohydrate oxidation was markedly depressed after diabetes in the presence of low (0.1 mM) but not high (1.0 mM) palmitate concentration. Increasing exogenous palmitate concentration 10-fold resulted in a 7-fold increase in the contribution of palmitate to energy production in controls but only a 30% increase in the diabetic group. Consequently, at 0.1 mM palmitate, the rate of fatty acid oxidation was higher in the diabetic group than in controls; however, at 1.0 mM fatty acid oxidation, it was significantly depressed. Therefore, after 1 wk of diabetes, the major differences in carbohydrate and fatty acid metabolism occur primarily at low rather than high exogenous palmitate concentration.

Lactic acid and protein interactions: implications for the NMR visibility of lactate in biological systems.

The addition of bovine serum albumin (BSA) to a solution of lactate and alanine resulted in the disappearance of the 1H-NMR resonances from lactate but not alanine. As temperature is increased lactate becomes increasingly NMR visible and after heating above 65 degreesC and cooling to 25 degreesC lactate binding is reduced. With a concentration of 0.2 mM BSA, there was a linear relationship between NMR visible lactate versus total lactate over a range of lactate concentrations of 0.2-35 mM (slope 0.384+/-0.003) indicating that approx. 60% of the added lactate is not visible in the 1H-NMR spectrum. With a 0.1 mM BSA solution, however, the slope was markedly higher indicating that under these conditions only 25-30% of the lactate was NMR invisible. The results from this study indicate that decreased NMR visibility of lactate in proteinaceous solutions is due to non-specific binding which is dependent on the tertiary structure of the protein. This has important implications not only for the interpretation of in vivo 1H-NMR experiments but also for 13C, and 14C studies of metabolism.

The effects of hypertrophy and diabetes on cardiac pyruvate dehydrogenase activity.

Alterations in substrate selection and utilisation are characteristics of heart failure of different etiologies and these changes may be involved in the development of contractile dysfunction. Regulation of pyruvate dehydrogenase (PDH) is crucial in determining the relative contribution of glucose oxidation to energy production; however, the role of PDH in the development of heart failure has not been clarified. In this study, we present a reliable and simple method for assaying both the active and total forms of PDH (PDHa and PDHt respectively) in cardiac tissue, and have compared the effects of pressure overload hypertrophy and diabetes on PDH activity. PDHa and PDHt were measured in extracts of hypertrophied hearts after 5 weeks of pressure overload or in hearts after 7 weeks following induction of diabetes. There was no significant change in PDHt in the hypertrophied group, but the fraction of PDH in the active form significantly decreased from 61+/-1% in controls to 36+/-1% (P<0.05). Following diabetes, there was a decrease in the ratio of PDHa:PDHt from 60+/-3% to 11+/-1% (P<0.0001) and PDHt activity -6.2+/-0.9 to 2.7+/-0.4 micromol/min/g wet weight (P<0.02)]. This study reports for the first time that (i) concomitant with the development of compensated hypertrophy, there is a decrease in the fraction of PDH in the active form; and (ii) in the diabetic heart, there is marked decrease in total PDH activity in addition to a decrease in the fraction of PDH in the active form. These results indicate that myocardial substrate delivery to the mitochondria may be impaired in both hypertrophy and diabetes, which may lead to the energy depleted state observed in heart failure.

Relationship between cardiac function and substrate oxidation in hearts of diabetic rats.

The effects of streptozotocin-induced diabetes on myocardial substrate oxidation and contractile function were investigated using 13C nuclear magnetic resonance (NMR) spectroscopy. To determine the consequences of diabetes on glucose oxidation, hearts were perfused with [1-13C]glucose (11 mM) alone as well as in the presence of insulin (to stimulate glucose transport) and dichloroacetate (to stimulate pyruvate dehydrogenase). The contribution of glucose to the tricarboxylic acid (TCA) cycle was significantly decreased in hearts from diabetic animals compared with controls, with glucose alone and with insulin; however, the addition of dichloroacetate significantly increased the contribution of glucose to the TCA cycle. Contractile function in hearts from diabetic animals was significantly depressed with glucose as the sole substrate, regardless of the presence of insulin or dichloroacetate (P < 0.0005). To determine whether diabetes had any direct effects on beta-oxidation and the TCA cycle, hearts were perfused with glucose (11 mM) plus [6-13C]hexanoate (0.5 mM) as substrates. In control hearts, with glucose plus hexanoate as substrates, hexanoate contributed 98.9 +/- 2% of the substrate entering the TCA cycle; this was significantly decreased to 90.7 +/- 0.6% in the diabetic group (P < 0.02). The addition of hexanoate to the perfusate resulted in a significant increase in peak systolic pressure in the diabetic group (P < 0.001) such that contractile function was indistinguishable from controls.

Acute L-triiodothyronine administration potentiates inotropic responses to beta-adrenergic stimulation in the isolated perfused rat heart.

OBJECTIVE: Acute inotropic effects of triiodothyronine (T3) have been reported, employing both in vivo experimental animal models and in vitro isolated heart perfusions. However, the mechanisms responsible for these acute inotropic effects remain unclear. The aim of this study, therefore, was to delineate the role of the beta-adrenergic receptor system in these acute responses. METHODS: The hearts from both euthyroid and hypothyroid (treated with 0.05% PTU in drinking water) male Sprague-Dawley rats were used in 5 experimental study protocols. Hearts from euthyroid rats were perfused with buffer containing either T3(10(-7) M) or control while continuously recording left ventricular function for 10 min ('acute effects'). Two-hour perfusions ('subacute effects') and cardiac responses following increasing doses of isoproterenol (10(-10) to 10(-6) M) in the presence or absence of T3-containing buffer (acute interaction) were also determined. In hypothyroid rats, the subacute responses and the acute interactions were investigated. RESULTS: In the presence of T3, an acute, significant potentiation of the inotropic responses following beta-adrenergic stimulation with isoproterenol was observed in both rat cohorts, which was more pronounced in hearts from euthyroid rats. An acute (< 40 s), but transient (79 +/- 8 s), direct inotropic response was observed in hearts from euthyroid rats. No cardiac responses were seen during a 2-h perfusion in hearts from either euthyroid or hypothyroid rats. CONCLUSIONS: The acute inotropic effects of T3 in non-ischemic myocardium probably result from an acute interaction between T3 and catecholamines rather than through a direct inotropic effect of T3 alone.

Metabolic compartmentation of lactate in the glucose-perfused rat heart.

Several studies using exogenous pyruvate as substrate have suggested that there are separate intracellular pyruvate pools in cardiac cells. Such heterogeneity in intracellular pyruvate has important implications for our understanding of both oxidative and nonoxidative glucose metabolism. Because pyruvate is not a major substrate for the heart in vivo, we wished to determine if there was pyruvate compartmentation with glucose as substrate. Hearts were isolated from male Sprague-Dawley rats and retrogradely perfused (Langendorff) with a modified Krebs-Henseleit buffer containing [1-13C[glucose for 5-115 min. At the end of each experiment, hearts were freeze-clamped and extracted for determination of the fractional enrichment of alanine, lactate, and acetyl CoA using 1H- and 13C-nuclear magnetic resonance spectroscopy. The fractional enrichment of alanine at the C-3 position was significantly higher than that for lactate at 55 min [43.6 +/- 2.8 vs. 27.2 +/- 4.6% (SD), n = 5, P < 0.003]. The differences in steady-state enrichment between lactate and alanine were not due to differences in the rate of labeling of these metabolites. The mean steady-state lactate enrichment was higher in the perfusate samples compared with the tissue samples from the same experiments (46.6 +/- 2.2 vs. 30.5 +/- 2.5%, n = 3, P < 0.005). Because fractional enrichment of alanine, acetyl CoA, and perfusate lactate are similar, we suggest that there is a separate nonexchanging pool of lactate rather than cytosolic compartmentation of pyruvate that has been previously proposed.

Cerebral metabolites in patients with acute and subacute strokes: concentrations determined by quantitative proton MR spectroscopy.

OBJECTIVE: The purpose of this study was to determine the feasibility of measuring concentrations of cerebral metabolites in acute and subacute stroke patients using single-voxel localized proton MR spectroscopy and to compare these concentrations to those in contralateral brain regions and in normal healthy volunteers. SUBJECTS AND METHODS: Single-voxel proton MR spectroscopy and MR imaging were performed in 14 stroke patients, at times ranging from 2 hr to 10 days following the onset of symptoms. Signals from choline, creatine, N-acetyl-L-aspartate (NAA), and lactate were quantified in the infarcted region (n = 14) and in the hemisphere contralateral to the stroke (n = 8) and compared with data obtained from a group of 10 control subjects. RESULTS: Infarcts were characterized by significantly increased lactate (12 of 14 patients; 7.5 +/- 8.9 mumol/g wet weight, mean +/- SD) and significantly decreased NAA (12 of 14 patients; 5.5 +/- 3.2 mumol/g wet weight), compared with contralateral brain regions and control data in healthy volunteers. Significant reductions in choline, creatine, and NAA were also found in contralateral brain regions compared with the control patients. CONCLUSION: Quantitative single-voxel proton spectroscopy is feasible for use in clinical studies of acute stroke. Ratio measurements or comparison with contralateral metabolites may be misleading because all metabolites may change during infarction, and contralateral metabolite levels may also be different from normal subjects.

Calculation of absolute metabolic flux and the elucidation of the pathways of glutamate labeling in perfused rat heart by 13C NMR spectroscopy and nonlinear least squares analysis.

Absolute metabolic fluxes in isolated perfused hearts have been determined by a nonlinear least squares analysis of glutamate labeling kinetics from [1-13C]glucose, [4-13C]beta-hydroxybutyrate, or [2-13C]acetate using 13C NMR spectroscopy. With glucose as substrate, the malate-aspartate shuttle flux was too slow to account for the reducing equivalents generated by glycolysis and to predict the observed oxygen consumption rate. For acetate and beta-hydroxybutyrate, the malate-aspartate shuttle had to be reversed for the network to agree with the observed oxygen consumption and glutamate labeling. Thus, an additional redox shuttle was required to reoxidize the NADH produced by cytoplasmic malate dehydrogenase. Using this model there was good agreement between the experimentally determined oxygen consumption and glutamate labeling and the calculated values of these parameters from the model for all substrates. The contribution of exogenous substrate to the overall tricarboxylic acid (TCA) cycle flux, 89.6 +/- 6.5% (mean +/- S.D.) as measured in the tissue extracts compared well with 91.4 +/- 4.2% calculated by the model. The ratio of TCA cycle flux to oxygen consumption for acetate, was 2.2 +/- 0.1, indicating that NADH production is principally accounted for by TCA cycle flux. For glucose or beta-hydroxybutyrate, this ratio was 2.9 +/- 0.2, consistent with the existence of other NADH producing reactions (e.g. glycolysis, beta-hydroxybutyrate oxidation).

Glucose metabolism in RIF-1 tumors after reduction in blood flow: an in vivo 13C and 31P NMR study.

Low pH appears to enhance the effectiveness of therapeutic hyperthermia. 13C and 31P NMR spectroscopy have been employed to examine the possibility that elevating glucose in a solid tumor while simultaneously reducing tumor blood flow would induce a more profound acidosis than either treatment alone. When blood flow in RIF-1 tumors was acutely reduced by administration of hydralazine and additional glucose was delivered locally by intratumoral injection, tumor acidosis (as determined by 31P NMR spectroscopy) during the period of reduced blood flow was not enhanced, relative to administration of hydralazine alone. Tumor NTP/P1 ratios decreased significantly within 20 min of hydralazine administration, whether or not glucose was injected, although NTP/P1 ratios were slightly higher in tumors that received extra glucose. Tumor lactate concentrations were not significantly different in glucose-supplemented tumors, despite glucose concentrations that were 4 to 5 times higher. When the added glucose was labeled with 13C, no correlation was detected between the pH in an individual tumor and the intensity of the 3-[13C]-lactate resonance in the same tumor.

Quantitative proton spectroscopy of canine brain: in vivo and in vitro correlations.

Quantitative, single-voxel proton NMR spectroscopy of normal brain was performed in five adult beagle dogs using the cerebral water signal as an internal intensity reference. The same brain regions were then rapidly isolated and frozen using a pneumatic biopsy drill, perchloric acid extracted, and analyzed by biochemical assay and high-resolution NMR spectroscopy. The concentrations of the major resonances in the in vivo and in vitro spectra were compared, and good agreement was found between the different measurements. The in vivo spectra contained three peaks at 3.21, 3.04, and 2.02 ppm, which are usually assigned to trimethylamines (TMA), creatines, and N-acetyl derivatives (NAc), which corresponded to be the following metabolite concentration values: 1.7 +/- 0.6, 7.7 +/- 2.1, and 10.9 +/- 2.7 mumol/g wet weight respectively. In vitro, the following metabolite concentrations were measured: glycerophosphocholine (GPC) 1.3 +/- 0.2, phosphocholine (PC) 0.5 +/- 0.1, phosphocreatine (PCr) 2.6 +/- 0.4, creatine (Cr) 5.9 +/- 1.4, and N-Acetyl aspartate (NAA) 8.9 +/- 1.8 mumol/g wet weight. Therefore, the 3.21 ppm resonance observed in the in vivo spectrum is predominantly GPC and PC in a ratio of 2.6:1, the 3.04 ppm resonance is Cr and PCr in a ratio of 2.3:1, and the 2.02 ppm resonance is predominantly (approximately 80%) NAA with small contributions from N-acetylaspartylglutamate (NAAG) and glutamate. The data presented here validate the technique of water referencing as a simple and convenient means of quantitating single-voxel in vivo proton NMR spectra of the brain.

A 13C-NMR study of glucose oxidation in the intact functioning rat heart following diabetes-induced cardiomyopathy.

A causative factor in the development of diabetes-induced heart dysfunction may be abnormalities in myocardial energy metabolism. Using 13C-NMR spectroscopy, we investigated the effects of experimentally induced diabetes (streptozotocin 65 mg/kg, i.v.) on glucose metabolism and contractile function in the isolated perfused rat heart. Hearts from streptozotocin-treated and untreated control rats were perfused with 11 mM [1-13C]glucose as substrate and 1H-decoupled 13C-spectra recorded for up to 90 min. Incorporation of label from [1-13C]glucose into lactate and glutamate was observed in hearts from control animals, consistent with metabolism through glycolysis and TCA cycle, respectively. Diabetic hearts did not incorporate label into lactate or glutamate. Addition of insulin (0.05 U/ml) to the buffer resulted in the appearance of [3-13C]lactate, although glutamate labeling was not observed. Addition of insulin plus dichloroacetate (2 mM) resulted in incorporation of label from [1-13C]glucose into 2-, 3- and 4-13C-glutamate, indicating glucose entry into the TCA cycle. Addition of insulin, or insulin plus dichloroacetate to control hearts did not alter labeling of either lactate or glutamate. Cardiac function in hearts from the diabetic group was depressed compared to controls and declined significantly over the duration of the experiment. These studies show that concomitant with a decrease in cardiac function, glucose oxidation is profoundly inhibited following the induction of diabetes with streptozotocin. These observations are consistent with a combination of decreased glucose transport and a decrease in pyruvate dehydrogenase activity.

Quantitative proton spectroscopy and histology of a canine brain tumor model.

Quantitative, single voxel proton nuclear magnetic resonance (NMR) spectroscopy and histological analysis was performed in eight dogs implanted with the transplantable canine glioma model of Wodinsky (Proc. Am. Assoc. Cancer Res. 10, 99 (1969)). Signals from choline, creatine, N-Acetyl Aspartate (NAA) and lactate were converted to molar concentration units and correlated with the quantitative analysis of histologically determined tissue types within the localized volume selected for NMR spectroscopy. In general, compared with normal brain, the lesions were associated with reductions in all metabolite concentrations, with the exception of lactate, which was increased. NAA and creatine decreases were most significantly correlated with the total lesion volume (P < 0.01), suggesting that these compounds are present in normal brain only. Changes in choline levels did not correlate strongly with any particular tissue type. Lactate was found to increase with increasing total lesion volume (P < 0.01), but not with increasing percent tumor, suggesting that it accumulates in abnormal tissue other than the tumor. The spectra reported were similar to those observed in human glioblastomas, with the exception that elevations of choline were not observed. The transplantable canine gliosarcoma system appears to be a suitable tumor model for evaluation by clinical radiological techniques such as magnetic resonance imaging (MRI) and proton NMR spectroscopy.