Compartmentation of glycolysis and glycogenolysis in the perfused rat heart

Developing methods that can detect compartmentation of metabolic pathways in intact tissues may be important for understanding energy demand and supply. In this study, we investigated compartmentation of glycolysis and glycogenolysis in the isolated perfused rat heart using (13)C NMR isotopomer analysis. Rat hearts previously depleted of myocardial glycogen were perfused with 5.5 mm [U-(13)C]glucose plus 50 mU/mL insulin until newly synthesized glycogen recovered to new steady-state levels ( approximately 60% of pre-depleted values). After a short wash-out period, the perfusate glucose was then switched to [1-(13)C]glucose, and glycolysis and glycogenolysis were stimulated by addition of glucagon (1 microg/ml). A (13)C NMR multiplet analysis of the methyl resonance of lactate provided an estimate of pyruvate derived from glucose vs glycogen while a multiplet analysis of the C4 resonance of glutamate provided an estimate of acetyl-CoA derived from glycolytic pyruvate vs glycogenolytic pyruvate. These two indices were not equivalent and their difference was further magnified in the presence of insulin during the stimulation phase. These combined observations are consistent with functional compartmentation of glycolytic and glycogenolytic enzymes that allows pyruvate generated by these two processes to be distinguished at the level of lactate and acetyl-CoA.     

Carbohydrate metabolism of the rat C6 glioma. An in vivo 13C and in vitro 1H magnetic resonance spectroscopy study

Surface coil 13C nuclear magnetic resonance (NMR) spectroscopy was used to investigate the in vivo carbohydrate metabolism of rat C6 gliomas during and after infusion with [1-13C] glucose. In vivo 1H-decoupled 13C NMR spectra of the glioma following infusion with [1-13C]glucose revealed the direct production of [3-13C]lactic acid, [1-13C]glycogen, and [4-13C], [3-13C], and [2-13C]glutamate/glutamine. Lactate levels of in vivo gliomas increased and reached steady state levels during [1-13C]glucose infusion, and decreased following termination of infusion. Complementary in vitro studies using supernatant media collected from C6 glioma cells incubated with media containing [1-13C] or [6-13C]glucose and glutamine were examined by 1H NMR spectroscopy. The [3-(13C/12C)]lactate ratios obtained from 1H spectra of supernatant media containing [1-13C]glucose revealed the percentage of glucose metabolized through the hexose monophosphate shunt to be 10.01 +/- 0.85% (n = 3), while similar measurements of media containing [6-13C]glucose and glutamine showed that glutaminolysis contributed 9.0 +/- 1.0% of total lactate production under these conditions. Enzymatic analysis of media determined lactate production to be 139 +/- 9 nmol per 10(6) cells per h (n = 4). These measurements demonstrate the ability of NMR to monitor brain tumor carbohydrate metabolism both in vitro and in vivo.     

Ketone bodies effectively compete with glucose for neuronal acetyl‐CoA generation in rat hippocampal slices

Ketone bodies can be used for cerebral energy generation in situ, when their availability is increased as during fasting or ingestion of a ketogenic diet. However, it is not known how effectively ketone bodies compete with glucose, lactate, and pyruvate for energy generation in the brain parenchyma. Hence, the contributions of exogenous 5.0 mM [1‐¹³C]glucose and 1.0 mM [2‐¹³C]lactate + 0.1 mM pyruvate (combined [2‐¹³C]lactate + [2‐¹³C]pyruvate) to acetyl‐CoA production were measured both without and with 5.0 mM [U‐¹³C]3‐hydroxybutyrate in superfused rat hippocampal slices by ¹³C NMR non‐steady‐state isotopomer analysis of tissue glutamate and GABA. Without [U‐¹³C]3‐hydroxybutyrate, glucose, combined lactate + pyruvate, and unlabeled endogenous sources contributed (mean ± SEM) 70 ± 7%, 10 ± 2%, and 20 ± 8% of acetyl‐CoA, respectively. With [U‐¹³C]3‐hydroxybutyrate, glucose contributions significantly fell from 70 ± 7% to 21 ± 3% (p < 0.0001), combined lactate + pyruvate and endogenous contributions were unchanged, and [U‐¹³C]3‐hydroxybutyrate became the major acetyl‐CoA contributor (68 ± 3%) – about three‐times higher than glucose. A direct analysis of the GABA carbon 2 multiplet revealed that [U‐¹³C]3‐hydroxybutyrate contributed approximately the same acetyl‐CoA fraction as glucose, indicating that it was less avidly oxidized by GABAergic than glutamatergic neurons. The appearance of superfusate lactate derived from glycolysis of [1‐¹³C]glucose did not decrease significantly in the presence of 3‐hydroxybutyrate, hence total glycolytic flux (Krebs cycle inflow + exogenous lactate formation) was attenuated by 3‐hydroxybutyrate. This indicates that, under these conditions, 3‐hydroxybutyrate inhibited glycolytic flux upstream of pyruvate kinase. Copyright © 2015 John Wiley & Sons, Ltd.     

Real‐time ALT and LDH activities determined in viable precision‐cut mouse liver slices using hyperpolarized [1‐13C]pyruvate—Implications for studies on biopsied liver tissues

Precision‐cut liver slices (PCLS) are widely used in liver research as they provide a liver model with all liver cell types in their natural architecture. The purpose of this study was to demonstrate the use of PCLS for hyperpolarized metabolic investigation in a mouse model, for potential future application in liver biopsy cores. Fresh normal liver was harvested from six mice. 500 μm PCLS were prepared and placed in a 10 mm NMR tube in an NMR spectrometer and perfused continuously. ³¹P spectra were acquired to evaluate the presence of adenosine triphosphate (ATP) and validate viability in all samples. Hyperpolarized [1‐¹³C]pyruvate was flushed into the NMR tube in the spectrometer. Consecutive ¹³C NMR spectra were acquired immediately after the injection using both non‐selective (five injections, two livers) and selective RF excitation (six injections, three livers). The ³¹P spectra showed the characteristic signals of ATP, confirming the viability of the PCLS for more than 2.5 h in the spectrometer. After each of the [1‐¹³C]pyruvate injections, both [1‐¹³C]lactate and [1‐¹³C]alanine signals were detected. Selective RF excitation aimed at both [1‐¹³C]lactate and [1‐¹³C]alanine enabled better visualization and quantification of the metabolic activity. Using this acquisition approach only the newly formed metabolites are observed upon excitation, and their intensities relative to those of hyperpolarized pyruvate enable quantification of metabolite production rates. This rate of lactate and alanine production appeared to be constant throughout the measurement time, with alanine production about 2.3 times higher than lactate. In summary, the viability of PCLS in an NMR spectrometer was demonstrated and hyperpolarized [1‐¹³C]pyruvate metabolism was recorded. This study opens up the possibility of evaluating alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activities in human liver biopsies, while preserving the tissue architecture and viability. In healthy, well‐perfused liver slices the ratio of ALT to LDH activity is about 2.3.     

A 13C NMR study on fluxes into the Krebs cycle of rabbit renal proximal tubular cells

Suspensions of rabbit renal proximal tubular (PCT) cells were incubated with [2-13C] and [3-13C]pyruvate. The perchloric acid extracts of the cell pellets were examined by 13C NMR. All experiments showed that enriched lactate, alanine, glutamate, and glutamine were the main metabolic intermediates, and that enrichment to a minor extent was found in the glutamate residue of glutathione (GSH). From these experiments, it could be deduced that PCT cells show a highly glycolytic activity, whereas enrichment of glucose exhibits gluconeogenesis. The estimation by 13C NMR of the ratio of the flux into the Krebs cycle via pyruvate carboxylase to the flux via pyruvate dehydrogenase is discussed. From incubations with 10 mM 13C-labelled pyruvate, we calculated from the relative enrichments of the glutamate carbon atoms that the ratio of pyruvate carboxylase to pyruvate dehydrogenase is 1.44 +/- 0.04 in rabbit renal proximal tubules.     

Effect of perfusate glucose concentration on rat lung glycolysis.

We investigated the relationship between perfusate concentration of glucose and its utilization and lactate production derived from exogenous glucose and from metabolism of endogenous substrates. Isolated rat lungs were ventilated with 5% CO2 in air and perfused for 100 min with Krebs-Ringer bicarbonate buffer containing 3% bovine serum albumin, 10(-2) U/ml insulin, [U-14C]glucose and [5-3H]glucose. Glucose utilization, total lactate production, [14C]lactate production, and 3H2O production were measured. The apparent Km and Vmax for glucose utilization were 3.4 mM and 72.5 mumol/g dry wt per h, respectively. Lactate production from endogenous substrates, calculated as the difference between total and [14C]lactate, was 37.6 +/- 2.2 mumol/g dry wt (n = 36); it was unaffected by perfusate glucose concentration and by omission of insulin, but increased threefold with anoxia. Lactate production from 1.5 mM glucose was significantly less (P less than 0.02) with insulin omitted. Glycogen content was unchanged during perfusion without glucose. These results suggest that: 1) protein catabolism contributes to lung lactate production; 2) glucose utilization by lung is not maximal at resting physiological glucose concentrations; and 3) insulin is required at low glucose concentrations for maximal glycolytic rates.     

Hepatic carbohydrate metabolism in the spontaneously diabetic Bio-Breeding Worcester rat

The effects of diabetes on hepatic carbohydrate metabolism were investigated in spontaneously diabetic Bio-Breeding Worcester (BB/W) rats. The juvenile-onset-type syndrome displayed by these animals is characterized by beta-cell destruction with subsequent ketosis-prone insulinopenia. Livers from diabetic animals demonstrated increased adenosine 3',5'-cyclic monophosphate levels but subnormal total protein and glycogen content. Isolated perfused livers of diabetic BB/W rats demonstrated an increased rate of glucose production from [14C]lactate and an impaired rate of glycogen synthesis. These data were consonant with hepatic enzyme studies demonstrating markedly increased activities of component gluconeogenic (glucose-6-phosphatase, fructose-1,6-diphosphatase, phosphoenolpyruvate carboxykinase) and glycogenolytic (glycogen phosphorylase) enzymes with decreased activities of glycolytic (hexokinase, pyruvate kinase) and glycogenic (glycogen synthase) enzymes. These findings agree with previous studies using alloxan- and streptozotocin-induced diabetic animals and suggest that accelerated hepatic gluconeogenesis and impaired glucose utilization are pathognomonic of all insulin-deficient diabetic syndromes.     

Hepatic metabolism of genetically diabetic (db/db) mice. I. Carbohydrate metabolism.

Hepatic carbohydrate metabolism in genetically diabetic mice (db/db) and their normal littermates has been studied. In db/db mice, body water was below normal and declined with age. The liver of db/db mice was abnormally large in relation to the metabolic mass of the body at all ages studied. In db/db mice, hepatic glycogenolysis, glycogen synthesis, glycogen synthetase, and phosphorylase were markedly increased. Gluconeogenesis from alanine or lactate in perfused livers of db/db mice was greater than normal per 100 g body water. Activities of fructose-1, 6-biophosphatase, glucose-6-phosphatase, glucokinase + hexokinase, and pyruvate kinase were elevated in livers of db/db mice. Diabetic mouse livers perfused with lactate showed a markedly reduced concentration of P-enolpyruvate and clear "forward crossover" between fructose-1, 6-P2 and fructose-6-P. In vivo glucose clearance, measured with [3-3H]glucose, in db/db mice was 170% that of normal mice. Data presented indicate that in livers of db/db mice: 1) glucose production is elevated prior to hyperglycemia, 2) glycogen turns over more rapidly, and 3) glycolytic and gluconeogenic enzymes are elevated paradoxically. These abnormalities are discussed from the viewpoint of their etiology.     

Metabolism of alternative substrates and the bioenergetic status of EMT6 tumor cell spheroids

In order to evaluate the ability of EMT6/Ro multicellular spheroids to utilize various pathways of energy production, (13)C and (31)P MRS have been employed to monitor the metabolism of glucose, glutamine, acetate and propionate. EMT6/Ro spheroids perfused with culture medium containing 5.5 mM glucose maintain stable levels of nucleotide triphosphates (NTP) and phosphocreatine (PCr) for up to 48 h, even in the absence of glutamine. The metabolism of 1-(13)C-glucose was almost entirely to 3-(13)C-lactate (88 +/- 12%, n = 7), even though the perfusion medium was equilibrated with 95% O(2). Labeling was also observed in other glycolytic metabolites, primarily alanine and alpha-glycerolphosphate. A low level of (13)C labeling in glutamate, indicative of mitochondrial oxidative metabolism (TCA cycle), was consistently detected when spheroids were perfused with 1-(13)C-glucose, almost exclusively in the C4 position of glutamate. Labeling of glutamate C2 and C3 was always less than 20% of the labeling in C4 and was usually undetectable. No evidence of adjacent carbon labeling in individual glutamate molecules (indicative of multiple cycles of label incorporation) was found, even in high-resolution (13)C NMR spectra of extracts from cells or spheroids. Despite the predominantly glycolytic metabolism of glucose, the mitochondrial substrate glutamine (2 mM, in the presence of < or =0.5 mM glucose from fetal bovine serum), supported stable levels of NTP and PCr in the tumor cells for up to 12 h. In the presence of 2.5 mM acetate, the bioenergetic status of cells in EMT6 spheroids declined slowly but measurably, and no incorporation of label from 2-(13)C-acetate into other metabolites was detected either in intact perfused spheroids or in high-resolution spectra of extracts. In contrast, when the anaplerotic TCA cycle substrate 3-(13)C-propionate replaced acetate, the high-energy phosphate levels in EMT6/Ro spheroids were somewhat reduced, but stabilized at a new lower level. Incubation of spheroids with 3-(13)C-propionate (with natural abundance glucose and glutamine) resulted in label detectable in the C2 and C3 of glutamate, but the primary labeled compound was methylmalonate, an intermediate in propionate metabolism. Addition of vitamin B(12), a cofactor for methylmalonyl CoA reductase, to the growth medium 24 h prior to perfusion with propionate resulted in the elimination of the methylmalonate resonance. A variety of 2- and 3-labeled metabolites were detected, including succinate, malate and glutamate. Labeling of C2 and C3 of lactate implicated cytoplasmic malic enzyme activity.     

Carbohydrate metabolism in perfused livers of adrenalectomized and steroid-replaced rats.

In livers from fasted rats perfused with bicarbonate buffer containing bovine albumin and erythrocytes, adrenalectomy decreased glycogen levels and glucose production, impaired the incorporation of 14C from [14C]lactate into glucose or glycogen, and decreased the activity of the active (I) form of glycogen synthase. Cortisol treatment restored gluconeogenesis after 1 h and glycogen synthesis after 2 h. Adrenalectomy did not alter the production of glucose or lactate or the levels of gluconeogenic intermediates in livers from fasted rats perfused with fructose, but reduced the formation of glycogen from this substrate. Adrenalectomy increased the levels of lactate and decreased the levels of P-pyruvate and subsequent intermediates in the gluconeogenic pathway. These changes were reversed by cortisol treatment. It is concluded that glucocorticoids support gluconeogenesis and glycogen synthesis in livers from fasted rats primarily by facilitating a reaction(s) located between pyruvate and P-pyruvate in the gluconeogenic pathway and by promoting the conversion of inactive to active glycogen synthase.     

Hyperpolarized 13C NMR studies of glucose metabolism in living breast cancer cell cultures

The recent development of dissolution dynamic nuclear polarization (DNP) gives NMR the sensitivity to follow metabolic processes in living systems with high temporal resolution. In this article, we apply dissolution DNP to study the metabolism of hyperpolarized U‐¹³C,²H₇‐glucose in living, perfused human breast cancer cells. Spectrally selective pulses were used to maximize the signal of the main product, lactate, whilst preserving the glucose polarization; in this way, both C₁‐lactate and C₃‐lactate could be observed with high temporal resolution. The production of lactate by T47D breast cancer cells can be characterized by Michaelis–Menten‐like kinetics, with K_m = 3.5 ± 1.5 mm and V_max = 34 ± 4 fmol/cell/min. The high sensitivity of this method also allowed us to observe and quantify the glycolytic intermediates dihydroxyacetone phosphate and 3‐phosphoglycerate. Even with the enhanced DNP signal, many other glycolytic intermediates could not be detected directly. Nevertheless, by applying saturation transfer methods, the glycolytic intermediates glucose‐6‐phosphate, fructose‐6‐phosphate, fructose‐1,6‐bisphosphate, glyceraldehyde‐3‐phosphate, phosphoenolpyruvate and pyruvate could be observed indirectly. This method shows great promise for the elucidation of the distinctive metabolism and metabolic control of cancer cells, suggesting multiple ways whereby hyperpolarized U‐¹³C,²H₇‐glucose NMR could aid in the diagnosis and characterization of cancer in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Robust glycogen shunt activity in astrocytes: Effects of glutamatergic and adrenergic agents

The significance and functional roles of glycogen shunt activity in the brain are largely unknown. It represents the fraction of metabolized glucose that passes through glycogen molecules prior to entering the glycolytic pathway. The present study was aimed at elucidating this pathway in cultured astrocytes from mouse exposed to agents such as a high [K⁺], d-aspartate and norepinephrine (NE) known to affect energy metabolism in response to neurotransmission. Glycogen shunt activity was assessed employing [1,6-¹³C]glucose, and the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) to block glycogen degradation. The label intensity in lactate, reflecting glycolytic activity, was determined by mass spectrometry. In the presence of NE a substantial glycogen shunt activity was observed, accounting for almost 40% of overall glucose metabolism. Moreover, when no metabolic stimulant was applied, a compensatory increase in glycolytic activity was seen when the shunt was inhibited by DAB. Actually the labeling in lactate exceeded that obtained when glycolysis and glycogen shunt both were operational, i.e. supercompensation. A similar phenomenon was seen when astrocytes were exposed to d-aspartate. In addition to glycolysis, tricarboxylic acid (TCA) cycle activity was monitored, analyzing labeling by mass spectrometry in glutamate which equilibrates with α-ketoglutarate. Both an elevated [K⁺] and d-aspartate induced an increased TCA cycle activity, which was altered when glycogen degradation was inhibited. Thus, the present study provides evidence that manipulation of glycogen metabolism affects both glycolysis and TCA cycle metabolism. Altogether, the results reveal a highly complex interaction between glycogenolysis and glycolysis, with the glycogen shunt playing a significant role in astrocytic energy metabolism.     

In a medium containing glucose, lactate carbon is incorporated by gonococci predominantly into fatty acids and glucose carbon incorporation is increased: implications regarding lactate stimulation of metabolism

The reason for stimulation by lactate of metabolism of gonococci growing in a medium containing glucose, which enhances pathogenicity by increasing growth rate, lipopolysaccharide (LPS) synthesis and protein formation, has been investigated. Tricine dodecylpolyacrylamide gel electrophoresis (SDS-PAGE) and thin layer chromatography (TLC) on homogenates of gonococci grown in this medium with [14C]lactate showed that lactate carbon was preferentially incorporated into lipid and LPS. Nuclear magnetic resonance (NMR) spectroscopy on lipid extracted from gonococci grown in the glucose containing medium with [13C]lactate showed that lactate carbon was incorporated into fatty acid moieties and not into ethanolamine or glycerol moieties. In contrast, NMR on lipid from gonococci grown with [13C]glucose indicated glucose carbon in both moieties. When unlabelled lactate was added, lipid synthesis from [l3C]glucose was stimulated and small amounts of different fatty acids were formed. The NMR data shows that gluconeogenesis from lactate carbon does not occur in the presence of glucose, suggesting that lactate is used solely for rapid production, via pyruvate, of acetyl CoA, the precursor not only for fatty acid synthesis but also for the constituents and products of the citric acid cycle, including ATP. The rapid formation of a high level of acetyl CoA is the probable reason for the stimulation of metabolism and oxygen uptake by lactate. 14C label on LPS was detected in its fatty acids. Most proteins that stained with silver in tricine SDS-PAGE were not significantly labelled by [14C]lactate in the glucose-containing medium. Two of three appreciably labelled proteins were identified by N-terminal sequencing as GroEL and porin 1B, and one of the two less labelled proteins was similar to peroxiredoxin type proteins. There were no signs of specific induction of these proteins by lactate and their labelling was consistent with fatty acids in attached lipid.     

Spatial heterogeneity of energy turnover in the heart

Local myocardial blood flow varies substantially in spite of a rather homogeneous morphology. To further elucidate this paradox, the spatial heterogeneity of tricarboxylic acid cycle turnover (J(TCA), micromol min(-1) g(-1)) and coronary flow was assessed at a high spatial resolution (6x6x6 mm3) in the open chest dog. Local flow differed more than 2.5-fold between individual samples in each heart (n=7). Out of 1,500 myocardial samples, 1/10 received less than 60% and another 1/10 more than 138% of the normalized mean. In low- and high-flow samples, pyruvate uptake and metabolism were analyzed by 13C NMR spectroscopy. Following [3-13C]pyruvate infusion (2 mM, 12 min), glutamate [4-13C]/[3-13C] was significantly greater in low-flow (2.21+/-0.75, 40 samples) than in high-flow (1.64+/-0.49, 39 samples) areas. This suggests that there are major differences in J(TCA). Glutamate, citrate and lactate content positively correlated with flow. Anaplerotic pathways contributed a fraction similar to J(TCA) in low- and high-flow areas, as demonstrated by isotopomer analysis after 60 min of [3-13C]pyruvate application. Mathematical model analysis of NMR data and relevant pool sizes revealed that J(TCA) and thus myocardial oxygen consumption (MVO2) in high-flow areas exceed values in low-flow areas at least threefold. Thus low and high metabolic states normally coexist within the well perfused heart, suggesting that there is considerable spatial heterogeneity of cardiac energy generation and work.     

Metabolism of [U‐13C]glucose in human brain tumors in vivo

Glioblastomas and brain metastases demonstrate avid uptake of 2‐[¹⁸F]fluoro‐2‐deoxyglucose by positron emission tomography and display perturbations of intracellular metabolite pools by ¹H MRS. These observations suggest that metabolic reprogramming contributes to brain tumor growth in vivo. The Warburg effect, excess metabolism of glucose to lactate in the presence of oxygen, is a hallmark of cancer cells in culture. 2‐[¹⁸F]Fluoro‐2‐deoxyglucose‐positive tumors are assumed to metabolize glucose in a similar manner, with high rates of lactate formation relative to mitochondrial glucose oxidation, but few studies have specifically examined the metabolic fates of glucose in vivo. In particular, the capacity of human brain cancers to oxidize glucose in the tricarboxylic acid cycle is unknown. Here, we studied the metabolism of human brain tumors in situ. [U‐¹³ C]Glucose (uniformly labeled glucose, i.e. d ‐glucose labeled with ¹³ C in all six carbons) was infused during surgical resection, and tumor samples were subsequently subjected to ¹³C NMR spectroscopy. The analysis of tumor metabolites revealed lactate production, as expected. We also determined that pyruvate dehydrogenase, turnover of the tricarboxylic acid cycle, anaplerosis and de novo glutamine and glycine synthesis contributed significantly to the ultimate disposition of glucose carbon. Surprisingly, less than 50% of the acetyl‐coenzyme A pool was derived from blood‐borne glucose, suggesting that additional substrates contribute to tumor bioenergetics. This study illustrates a convenient approach that capitalizes on the high information content of ¹³C NMR spectroscopy and enables the analysis of intermediary metabolism in diverse cancers growing in their native microenvironment. Copyright © 2012 John Wiley & Sons, Ltd.     

Pyruvate‐lactate exchange and glucose uptake in human prostate cancer cell models. A study in xenografts and suspensions by hyperpolarized [1‐13C]pyruvate MRS and [18F]FDG‐PET

Reprogramming of energy metabolism in the development of prostate cancer can be exploited for a better diagnosis and treatment of the disease. The goal of this study was to determine whether differences in glucose and pyruvate metabolism of human prostate cancer cells with dissimilar aggressivenesses can be detected using hyperpolarized [1‐¹³C]pyruvate MRS and [¹⁸F]FDG‐PET imaging, and to evaluate whether these measures correlate. For this purpose, we compared murine xenografts of human prostate cancer LNCaP cells with those of more aggressive PC3 cells. [1‐¹³C]pyruvate was hyperpolarized by dissolution dynamic nuclear polarization (dDNP) and [1‐¹³C]pyruvate to lactate conversion was followed by ¹³C MRS. Subsequently [¹⁸F]FDG uptake was investigated by static and dynamic PET measurements. Standard uptake values (SUVs) for [¹⁸F]FDG were significantly higher for xenografts of PC3 compared with those of LNCaP. However, we did not observe a difference in the average apparent rate constant k_pl of ¹³C label exchange from pyruvate to lactate between the tumor variants. A significant negative correlation was found between SUVs from [¹⁸F]FDG PET measurements and k_pl values for the xenografts of both tumor types. The k_pl rate constant may be influenced by various factors, and studies with a range of prostate cancer cells in suspension suggest that LDH inhibition by pyruvate may be one of these. Our results indicate that glucose and pyruvate metabolism in the prostate cancer cell models differs from that in other tumor models and that [¹⁸F]FDG‐PET can serve as a valuable complementary tool in dDNP studies of aggressive prostate cancer with [1‐¹³C]pyruvate.     

Involvement of brain lactate in neuronal metabolism

The involvement of brain lactate in neuronal metabolism was analyzed by ex vivo NMR spectroscopy with rats under the effects of pentobarbital, alphachloralose or morphine, which were infused with a solution of either [1-(13)C]glucose+lactate or glucose+[3-(13)C]lactate for 20 min. Electroencephalogram recordings indicated different brain electrical activity levels under the three drugs with a clear distinction between pentobarbital, on the one hand, and alphachloralose and morphine on the other. Labeling of metabolites in brain perchloric acid extracts and of blood glucose and lactate was determined by (13)C- and/or (1)H-observed/(13)C-edited-NMR spectroscopy. The following were found: (i) the ratio between glutamate C3 and C4 (13)C-enrichments increased from pentobarbital to alphachloralose and morphine whatever the labeled precursor, indicating a link between metabolic and electrical activity; (ii) under glucose+[3-(13)C]lactate infusion, alanine C3 and acetyl-CoA C2 enrichments were higher than that of lactate C3, revealing the occurrence of an isotopic dilution of the brain exogenous lactate (arising from blood) by lactate from brain (endogenous lactate); the latter was synthesized from glycolysis in a compartment other than the neurons; (iii) the contributions of labeled glucose and lactate to acetyl-CoA C2 enrichment indicated that the involvement of blood glucose relative to that of blood lactate to brain metabolism was correlated with brain activity. It can therefore be concluded that the brain electrical activity-dependent increase in the contribution of blood glucose relative to that of blood lactate to brain metabolism occurred partly via the increase in the metabolism of lactate generated from astrocytic glycolysis. This conclusion supports the hypothesis of an astrocyte-neuron lactate shuttle component in the coupling mechanism between cerebral activity and energy metabolism.     

Influence of altered O2 tension on substrate metabolism in perfused rat lung.

Effects of hypoxia (1.5 h) on glucose and palmitate metabolism were investigated in perfused lungs from normal rats and rats exposed for 24 h to hypobaric conditions (simulated altitude of 24,000 ft). Hypoxic lungs were ventilated with 5% O2-5% CO2 and control lungs with 21% O2-5% CO2. Blood gases and pH remained stable during the 1.5-h perfusion period. Exposure of normal rat lungs to 1.5 h of in vitro hypoxia (blood Po2=34 mmHg) significantly increased lactate production and mean arterial pulmonary pressure, but did not alter glucose uptake, pyruvate levels, and oxidation of either [U-14C]glucose or [1-14C]palmitate to CO2. Incorporation of labeled glucose and palmitate into lung lipids was also unaltered. In contrast to normal lungs, prior exposure to hypoxia for 24 h and subsequent perfusion under hypoxic conditions significantly stimulated glucose uptake (74% increase), markedly increased glucose incorporation into lung lipids, and increased oxidation of glucose to CO2. Lactate/pyruvate ratios also showed a significant 38% increase. Lung glycogen was unchanged following 24 h hypoxia. These data indicate that adaptive changes occur in metabolic processes within the lung during acute changes in O2 tension.     

Substrate selection in hearts subjected to ischemia/reperfusion: role of cardioplegic solutions and gender

In conditions of ischemia/reperfusion (I/R), the relative use of all available substrates by the heart has a significant effect on the recovery of the organ. This substrate preference in perfused hearts is influenced by ischemia. We followed the metabolic fate of [U‐¹³C]glucose and [3‐¹³C]lactate in hearts preserved in Celsior (Cs) and histidine buffer solution (HBS) for 4 or 6 h and subsequently perfused with a Krebs–Henseleit solution (KH) containing [U‐¹³C]glucose and [3‐¹³C]lactate. We also assessed gender‐specific metabolic modulation in our I/R experimental conditions. Hearts from male and female Wistar rats (6–8 weeks) were subjected to moderate (0–240 min) or prolonged (240–360 min) cold ischemia whilst immersed in Cs and HBS, and perfused for 30 min with KH containing [U‐¹³C]glucose and [3‐¹³C]lactate. After perfusion, hearts were freeze‐clamped and metabolites were extracted for ¹³C NMR isotopomer analysis. In control conditions, there were no differences with regard to lactate origin in hearts from males and females. After 6 h of preservation in Cs, lactate origin was mostly from [U‐¹³C]glucose in hearts from males and from [3‐¹³C]lactate in hearts from females. During the 6 h of organ preservation in HBS, the lactate pool showed a strong contribution from unenriched sources in male hearts and from [U‐¹³C]glucose in female hearts. The glutamate C2/C4 ratio was stable or increased in hearts from females after I/R, and the alanine index increased in hearts from both males and females. Octanoate was, as predicted, the preferential substrate during perfusion. Glucose and lactate suffer a distinct metabolic fate in our I/R conditions, which is related to the cardioplegic solution used during organ storage, and the gender. Hearts from females appear to be less sensitive to I/R injury, and heart preservation in HBS proved to be effective in enhancing anaplerosis during perfusion, especially in hearts from females. Copyright © 2011 John Wiley & Sons, Ltd.