Effect of 2-deoxy-d-Glucose on Aminoacids Metabolism in Rats’ Cerebral Cortex Slices

We studied the effect of different concentrations of 2-deoxy-d-glucose on the l-[U-¹⁴C]leucine, l-[1-¹⁴C]leucine and [1-¹⁴C]glycine metabolism in slices of cerebral cortex of 10-day-old rats. 2-deoxy-d-glucose since 0.5 mM concentration has inhibited significantly the protein synthesis from l-[U-¹⁴C]leucine and from [1-¹⁴C]glycine in relation to the medium containing only Krebs Ringer bicarbonate. Potassium 8.0 mM in incubation medium did not stimulate the protein synthesis compared to the medium containing 2.7 mM, and at 50 mM diminishes more than 2.5 times the protein synthesis compared to the other concentration. Only at the concentration of 5.0 mM, 2-deoxy-d-glucose inhibited the CO₂ production and lipid synthesis from l-[U-¹⁴C] leucine. This compound did not inhibit either CO₂ production, or lipid synthesis from [1-¹⁴C]glycine. Lactate at 10 mM and glucose 5.0 mM did not revert the inhibitory effect of 2-deoxy-d-glucose on the protein synthesis from l-[U-¹⁴C]leucine. 2-deoxy-d-glucose at 2.0 mM did not show any effect either on CO₂ production, or on lipid synthesis from l-[U-¹⁴C]lactate 10 mM and glucose 5.0 mM.     

Evidence for transketolase-like TKTL1 flux in CHO cells based on parallel labeling experiments and 13C-metabolic flux analysis

The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. It provides precursors for the biosynthesis of nucleotides and contributes to the production of reducing power in the form of NADPH. It has been hypothesized that mammalian cells may contain a hidden reaction in PPP catalyzed by transketolase-like protein 1 (TKTL1) that is closely related to the classical transketolase enzyme; however, until now there has been no direct experimental evidence for this reaction. In this work, we have applied state-of-the-art techniques in ¹³C metabolic flux analysis (¹³C-MFA) based on parallel labeling experiments and integrated flux fitting to estimate the TKTL1 flux in CHO cells. We identified a set of three parallel labeling experiments with [1-¹³C]glucose+[4,5,6-¹³C]glucose, [2-¹³C]glucose+[4,5,6-¹³C]glucose, and [3-¹³C]glucose+[4,5,6-¹³C]glucose and developed a new method to measure ¹³C-labeling of fructose 6-phosphate by GC-MS that allows intuitive interpretation of mass isotopomer distributions to determine key fluxes in the model, including glycolysis, oxidative PPP, non-oxidative PPP, and the TKTL1 flux. Using these tracers we detected a significant TKTL1 flux in CHO cells at the stationary phase. The flux results suggest that the main function of oxidative PPP in CHO cells at the stationary phase is to fuel the TKTL1 reaction. Overall, this study demonstrates for the first time that carbon atoms can be lost in the PPP, by means other than the oxidative PPP, and that this loss of carbon atoms is consistent with the hypothesized TKTL1 reaction in mammalian cells.     

Stable isotope-resolved metabolomic analysis of lithium effects on glial-neuronal metabolism and interactions

Despite the long-established therapeutic efficacy of lithium in the treatment of bipolar disorder (BPD), its molecular mechanism of action remains elusive. Newly developed stable isotope-resolved metabolomics (SIRM) is a powerful approach that can be used to elucidate systematically how lithium impacts glial and neuronal metabolic pathways and activities, leading ultimately to deciphering its molecular mechanism of action. The effect of lithium on the metabolism of three different ¹³C-labeled precursors ([U-¹³C]-glucose, ¹³C-3-lactate or ¹³C-2,3-alanine) was analyzed in cultured rat astrocytes and neurons by nuclear magnetic resonance (NMR) spectroscopy and gas chromatography mass spectrometry (GC-MS). Using [U-¹³C]-glucose, lithium was shown to enhance glycolytic activity and part of the Krebs cycle activity in both astrocytes and neurons, particularly the anaplerotic pyruvate carboxylation (PC). The PC pathway was previously thought to be active in astrocytes but absent in neurons. Lithium also stimulated the extracellular release of ¹³C labeled-lactate, -alanine (Ala), -citrate, and -glutamine (Gln) by astrocytes. Interrogation of neuronal pathways using ¹³C-3-lactate or ¹³C-2,3-Ala as tracers indicated a high capacity of neurons to utilize lactate and Ala in the Krebs cycle, particularly in the production of labeled Asp and Glu via PC and normal cycle activity. Prolonged lithium treatment enhanced lactate metabolism via PC but inhibited lactate oxidation via the normal Krebs cycle in neurons. Such lithium modulation of glycolytic, PC and Krebs cycle activity in astrocytes and neurons as well as release of fuel substrates by astrocytes should help replenish Krebs cycle substrates for Glu synthesis while meeting neuronal demands for energy. Further investigations into the molecular regulation of these metabolic traits should provide new insights into the pathophysiology of mood disorders and early diagnostic markers, as well as new target(s) for effective therapies.     

Stable isotope resolved metabolomics analysis of ribonucleotide and RNA metabolism in human lung cancer cells

We have developed a simple NMR-based method to determine the turnover of nucleotides and incorporation into RNA by stable isotope resolved metabolomics (SIRM) in A549 lung cancer cells. This method requires no chemical degradation of the nucleotides or chromatography. During cell growth, the free ribonucleotide pool is rapidly replaced by de novo synthesized nucleotides. Using [U-¹³C]-glucose and [U-¹³C,¹⁵N]-glutamine as tracers, we showed that virtually all of the carbons in the nucleotide riboses were derived from glucose, whereas glutamine was preferentially utilized over glucose for pyrimidine ring biosynthesis, via the synthesis of Asp through the Krebs cycle. Incorporation of the glutamine amido nitrogen into the N3 and N9 positions of the purine rings was also demonstrated by proton-detected ¹⁵N NMR. The incorporation of ¹³C from glucose into total RNA was measured and shown to be a major sink for the nucleotides during cell proliferation. This method was applied to determine the metabolic action of an anti-cancer selenium agent (methylseleninic acid or MSA) on A549 cells. We found that MSA inhibited nucleotide turnover and incorporation into RNA, implicating an important role of nucleotide metabolism in the toxic action of MSA on cancer cells.     

Interaction between the Pentose Phosphate Pathway and Gluconeogenesis from Glycerol in the Liver

After exposure to [U-¹³C₃]glycerol, the liver produces primarily [1,2,3-¹³C₃]- and [4,5,6-¹³C₃]glucose in equal proportions through gluconeogenesis from the level of trioses. Other ¹³C-labeling patterns occur as a consequence of alternative pathways for glucose production. The pentose phosphate pathway (PPP), metabolism in the citric acid cycle, incomplete equilibration by triose phosphate isomerase, or the transaldolase reaction all interact to produce complex ¹³C-labeling patterns in exported glucose. Here, we investigated ¹³C labeling in plasma glucose in rats given [U-¹³C₃]glycerol under various nutritional conditions. Blood was drawn at multiple time points to extract glucose for NMR analysis. Because the transaldolase reaction and incomplete equilibrium by triose phosphate isomerase cannot break a ¹³C-¹³C bond within the trioses contributing to glucose, the appearance of [1,2-¹³C₂]-, [2,3-¹³C₂]-, [5,6-¹³C₂]-, and [4,5-¹³C₂]glucose provides direct evidence for metabolism of glycerol in the citric acid cycle or the PPP but not an influence of either triose phosphate isomerase or the transaldolase reaction. In all animals, [1,2-¹³C₂]glucose/[2,3-¹³C₂]glucose was significantly greater than [5,6-¹³C₂]glucose/[4,5-¹³C₂]glucose, a relationship that can only arise from gluconeogenesis followed by passage of substrates through the PPP. In summary, the hepatic PPP in vivo can be detected by ¹³C distribution in blood glucose after [U-¹³C₃]glycerol administration.     

Single valproic acid treatment inhibits glycogen and RNA ribose turnover while disrupting glucose-derived cholesterol synthesis in liver as revealed by the [U-13C6]-d-glucose tracer in mice

Previous genetic and proteomic studies identified altered activity of various enzymes such as those of fatty acid metabolism and glycogen synthesis after a single toxic dose of valproic acid (VPA) in rats. In this study, we demonstrate the effect of VPA on metabolite synthesis flux rates and the possible use of abnormal ¹³C labeled glucose-derived metabolites in plasma or urine as early markers of toxicity. Female CD-1 mice were injected subcutaneously with saline or 600 mg/kg) VPA. Twelve hours later, the mice were injected with an intraperitoneal load of 1 g/kg [U-¹³C]-d-glucose. ¹³C isotopomers of glycogen glucose and RNA ribose in liver, kidney and brain tissue, as well as glucose disposal via cholesterol and glucose in the plasma and urine were determined. The levels of all of the positional ¹³C isotopomers of glucose were similar in plasma, suggesting that a single VPA dose does not disturb glucose absorption, uptake or hepatic glucose metabolism. Three-hour urine samples showed an increase in the injected tracer indicating a decreased glucose re-absorption via kidney tubules. ¹³C labeled glucose deposited as liver glycogen or as ribose of RNA were decreased by VPA treatment; incorporation of ¹³C via acetyl-CoA into plasma cholesterol was significantly lower at 60 min. The severe decreases in glucose-derived carbon flux into plasma and kidney-bound cholesterol, liver glycogen and RNA ribose synthesis, as well as decreased glucose re-absorption and an increased disposal via urine all serve as early flux markers of VPA-induced adverse metabolic effects in the host.     

A simple method for determining total glucose utilisation by isolated adipocytes using (5-3H)-glucose

Glucose utilisation by adipocytes incubated with and without insulin and at two concentrations of extracellular glucose has been estimated by three different procedures. Glucose disappearance from the medium was calculated by using glucose oxidase to determine the glucose concentration remaining after incubation and comparing this with the glucose concentration in standard solutions made up by appropriate dilution of the original medium. [U-¹⁴C]-glucose utilisation was calculated by summing the ¹⁴C found in CO₂, triglycerides, and anions. [³H]-H₂O formation from [5-³H]-glucose was the third measure of glucose utilisation. All three methods gave similar answers, but the [5-³H]-glucose is simpler to use than [U-¹⁴C]-glucose and gives substantially more reproducible results than glucose oxidase.     

Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by 13C metabolic flux analysis

Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli, until now there have been only a handful of studies focused on anaerobic glucose metabolism and no ¹³C-flux studies on xylose metabolism. In the absence of experimentally validated flux maps, constraint-based approaches such as MOMA and RELATCH cannot be used to guide new metabolic engineering designs. In this work, we have addressed this critical gap in current understanding by performing comprehensive characterizations of glucose and xylose metabolism under aerobic and anaerobic conditions, using recent state-of-the-art techniques in ¹³C metabolic flux analysis (¹³C-MFA). Specifically, we quantified precise metabolic fluxes for each condition by performing parallel labeling experiments and analyzing the data through integrated ¹³C-MFA using the optimal tracers [1,2-¹³C]glucose, [1,6-¹³C]glucose, [1,2-¹³C]xylose and [5-¹³C]xylose. We also quantified changes in biomass composition and confirmed turnover of macromolecules by applying [U-¹³C]glucose and [U-¹³C]xylose tracers. We demonstrated that under anaerobic growth conditions there is significant turnover of lipids and that a significant portion of CO₂ originates from biomass turnover. Using knockout strains, we also demonstrated that β-oxidation is critical for anaerobic growth on xylose. Quantitative analysis of co-factor balances (NADH/FADH₂, NADPH, and ATP) for different growth conditions provided new insights regarding the interplay of energy and redox metabolism and the impact on E. coli cell physiology.     

Carbohydrate oxidation by leucoplasts from suspension cultures of soybean (Glycine max L.)

The aim of this work was to discover how leucoplasts from suspension cultures of soybean (Glycine max L.) oxidize hexose monophosphates. Leucoplasts were isolated from protoplast lysates on a continuous gradient of Nycodenz with a yield of 28% and an intactness of 80%. Incubation of the leucoplasts with ¹⁴C-labelled substrates led to ¹⁴CO₂ production, that was dependent upon leucoplast intactness, from [U-¹⁴C]glucose 6-phosphate, [U-¹⁴C]glucose 1-phosphate, [U-¹⁴C] fructose 6-phosphate and [U-¹⁴C]glucose+ATP, but not from [U-¹⁴C]fructose-1,6-bisphosphate or [U-¹⁴C]triose phosphate. The yield from [U-¹⁴C]glucose 6-phosphate was at least four times greater than that from any of the other substrates. When [1-¹⁴C]-, [2-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose 6-phosphate were supplied to leucoplasts significant ¹⁴CO₂ production that was dependent upon leucoplast intactness was found only for [1-¹⁴C]glucose 6-phosphate. It is argued that soybean cell leucoplasts oxidize glucose 6-phosphate via the oxidative pentose phosphate pathway with very little recycling, and that in these plastids glycolysis to acetyl CoA is negligible.S.A.C. thanks the Science and Engineering Research Council for a research studentship.     

Cytosolic NADPH Homeostasis in Glucose-starved Procyclic Trypanosoma brucei Relies on Malic Enzyme and the Pentose Phosphate Pathway Fed by Gluconeogenic Flux

All living organisms depend on NADPH production to feed essential biosyntheses and for oxidative stress defense. Protozoan parasites such as the sleeping sickness pathogen Trypanosoma brucei adapt to different host environments, carbon sources, and oxidative stresses during their infectious life cycle. The procyclic stage develops in the midgut of the tsetse insect vector, where they rely on proline as carbon source, although they prefer glucose when grown in rich media. Here, we investigate the flexible and carbon source-dependent use of NADPH synthesis pathways in the cytosol of the procyclic stage. The T. brucei genome encodes two cytosolic NADPH-producing pathways, the pentose phosphate pathway (PPP) and the NADP-dependent malic enzyme (MEc). Reverse genetic blocking of those pathways and a specific inhibitor (dehydroepiandrosterone) of glucose-6-phosphate dehydrogenase together established redundancy with respect to H₂O₂ stress management and parasite growth. Blocking both pathways resulted in ∼10-fold increase of susceptibility to H₂O₂ stress and cell death. Unexpectedly, the same pathway redundancy was observed in glucose-rich and glucose-depleted conditions, suggesting that gluconeogenesis can feed the PPP to provide NADPH. This was confirmed by (i) a lethal phenotype of RNAi-mediated depletion of glucose-6-phosphate isomerase (PGI) in the glucose-depleted Δmec/Δmec null background, (ii) an ∼10-fold increase of susceptibility to H₂O₂ stress observed for the Δmec/Δmec/^RNAiPGI double mutant when compared with the single mutants, and (iii) the ¹³C enrichment of glycolytic and PPP intermediates from cells incubated with [U-¹³C]proline, in the absence of glucose. Gluconeogenesis-supported NADPH supply may also be important for nucleotide and glycoconjugate syntheses in the insect host.     

A metabolic flux analysis to study the role of sucrose synthase in the regulation of the carbon partitioning in central metabolism in maize root tips

In order to understand the role of sucrose synthase (SuSy) in carbon partitioning, metabolic fluxes were analyzed in maize root tips of a double mutant of SuSy genes, sh1 sus1 and the corresponding wild type, W22. [U-¹⁴C]-glucose pulse labeling experiments permitted the quantification of unidirectional fluxes into sucrose, starch and cell wall polysaccharides. Isotopic steady-state labeling with [1-¹³C]-, [2-¹³C]- or [U-¹³C]-glucose followed by the quantification by ¹H-NMR and ¹³C-NMR of enrichments in carbohydrates and amino acids was also performed to determine 29 fluxes through central metabolism using computer-aided modeling. As a consequence of the suppression of SUS1 and SH1 isozymes, maize root tips diameter was significantly decreased and respiratory metabolism reduced by 30%. Our result clearly established that, in maize root tips, starch is produced from ADP-Glc synthesized in the plastid and not in the cytosol by sucrose synthase. Unexpectedly, the flux of cell wall synthesis was increased in the double mutant. This observation indicates that, in maize root tips, SH1 and SUS1 are not specific providers for cellulose biosynthesis.     

Dynamic Measurements of Cerebral Pentose Phosphate Pathway Activity In Vivo Using [1,6-13C2,6,6-2H2] Glucose and Microdialysis

Abstract: Cerebral pentose phosphate pathway (PPP) activity has been linked to NADPH-dependent anabolic pathways, turnover of neurotransmitters, and protection from oxidative stress. Research on this potentially important pathway has been hampered, however, because measurement of regional cerebral PPP activity in vivo has not been possible. Our efforts to address this need focused on the use of a novel isotopically substituted glucose molecule, [1,6-¹³C₂,6,6-²H₂]glucose, in conjunction with microdialysis techniques, to measure cerebral PPP activity in vivo, in freely moving rats. Metabolism of [1,6-¹³C₂,6,6-²H₂]glucose through glycolysis produces [3-¹³C]lactate and [3-¹³C,3,3-²H₂]lactate, whereas metabolism through the PPP produces [3-¹³C,3,3-²H₂]lactate and unlabeled lactate. The ratios of these lactate isotopomers can be quantified using gas chromatography/mass spectrometry (GC/MS) for calculation of PPP activity, which is reported as the percentage of glucose metabolized to lactate that passed through the PPP. Following addition of [1,6-¹³C₂,6,6-²H₂]glucose to the perfusate, labeled lactate was easily detectable in dialysate using GC/MS. Basal forebrain and intracerebral 9L glioma PPP values (mean ± SD) were 3.5 ± 0.4 (n = 4) and 6.2 ± 0.9% (n = 4), respectively. Furthermore, PPP activity could be stimulated in vivo by addition of phenazine methosulfate, an artificial electron acceptor for NADPH, to the perfusion stream. These results show that the activity of the PPP can now be measured dynamically and regionally in the brains of conscious animals in vivo.     

Metabolic Compartmentation in Cortical Synaptosomes: Influence of Glucose and Preferential Incorporation of Endogenous Glutamate into GABA

Metabolism of glutamine was determined under a variety of conditions to study compartmentation in cortical synaptosomes. The combined intracellular and extracellular amounts of [U-¹³C]GABA, [U-¹³C]glutamate and [U-¹³C]glutamine were the same in synaptosomes incubated with [U-¹³C]glutamine in the presence and absence of glucose. However, the concentration of these amino acids was decreased in the latter group, demonstrating the requirement for glucose to maintain the size of neurotransmitter pools. In hypoglycemic synaptosomes more [U-¹³C]glutamine was converted to [U-¹³C]aspartate, and less glutamate was re-synthesized from the tricarboxylic acid (TCA) cycle, suggesting use of the partial TCA cycle from -ketoglutarate to oxaloacetate for energy. Compartmentation was studied in synaptosomes incubated with glucose plus labeled and unlabeled glutamine and glutamate. Incubation with [U-¹³C]glutamine plus unlabeled glutamate gave rise to [U-¹³C]GABA but not labeled aspartate; however, incubation with [U-¹³C]glutamate plus unlabeled glutamine gave rise to [U-¹³C]aspartate, but not labeled GABA. Thus the endogenous glutamate formed via glutaminase in synaptic terminals is preferentially used for GABA synthesis, and is metabolized differently than glutamate taken up from the extracellular milieu.     

Selective <Superscript>1</Superscript>H-<Superscript>13</Superscript>C NMR spectroscopy of methyl groups in residually protonated samples of large proteins

Methyl ¹³CHD₂ isotopomers of all methyl-containing amino-acids can be observed in residually protonated samples of large proteins obtained from [U-¹³C,¹H]-glucose/D₂O-based bacterial media, with sensitivity sufficient for a number of NMR applications. Selective detection of some subsets of methyl groups (Ala^β, Thr^γ2) is possible using simple ‘out-and-back’ NMR methodology. Such selective methyl-detected ‘out-and-back’ NMR experiments allow complete assignments of threonine γ2 methyls in residually protonated, [U-¹³C,¹H]-glucose/D₂O-derived samples of an 82-kDa enzyme Malate Synthase G. [U-¹³C,¹H]-glucose/D₂O-derived protein samples are relatively inexpensive and are usually available at very early stages of any NMR study of high-molecular-weight systems.     

Integrated 13C-metabolic flux analysis of 14 parallel labeling experiments in Escherichia coli

The use of parallel labeling experiments for ¹³C metabolic flux analysis (¹³C-MFA) has emerged in recent years as the new gold standard in fluxomics. The methodology has been termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. In this contribution, we have tested the limits of COMPLETE-MFA by demonstrating integrated analysis of 14 parallel labeling experiments with Escherichia coli. An effort on such a massive scale has never been attempted before. In addition to several widely used isotopic tracers such as [1,2-¹³C]glucose and mixtures of [1-¹³C]glucose and [U-¹³C]glucose, four novel tracers were applied in this study: [2,3-¹³C]glucose, [4,5,6-¹³C]glucose, [2,3,4,5,6-¹³C]glucose and a mixture of [1-¹³C]glucose and [4,5,6-¹³C]glucose. This allowed us for the first time to compare the performance of a large number of isotopic tracers. Overall, there was no single best tracer for the entire E. coli metabolic network model. Tracers that produced well-resolved fluxes in the upper part of metabolism (glycolysis and pentose phosphate pathways) showed poor performance for fluxes in the lower part of metabolism (TCA cycle and anaplerotic reactions), and vice versa. The best tracer for upper metabolism was 80% [1-¹³C]glucose+20% [U-¹³C]glucose, while [4,5,6-¹³C]glucose and [5-¹³C]glucose both produced optimal flux resolution in the lower part of metabolism. COMPLETE-MFA improved both flux precision and flux observability, i.e. more independent fluxes were resolved with smaller confidence intervals, especially exchange fluxes. Overall, this study demonstrates that COMPLETE-MFA is a powerful approach for improving flux measurements and that this methodology should be considered in future studies that require very high flux resolution.     

Stable isotope resolved metabolomics of lung cancer in a SCID mouse model

We have determined the time course of [U-¹³C]-glucose utilization and transformations in SCID mice via bolus injection of the tracer in the tail vein. Incorporation of ¹³C into metabolites extracted from mouse blood plasma and several tissues (lung, heart, brain, liver, kidney, and skeletal muscle) were profiled by NMR and GC–MS, which helped ascertain optimal sampling times for different target tissues. We found that the time for overall optimal ¹³C incorporation into tissue was 15–20 min but with substantial differences in ¹³C labeling patterns of various organs that reflected their specific metabolism. Using this stable isotope resolved metabolomics (SIRM) approach, we have compared the ¹³C metabolite profile of the lungs in the same mouse with or without an orthotopic lung tumor xenograft established from human PC14PE6 lung adenocarcinoma cells. The ¹³C metabolite profile shows considerable differences in [U-¹³C]-glucose transformations between the two lung tissues, demonstrating the feasibility of applying SIRM to investigate metabolic networks of human cancer xenograft in the mouse model.     

Metabolism of [U-<Superscript>13</Superscript>C]Glutamine and [U-<Superscript>13</Superscript>C]Glutamate in Isolated Rat Brain Mitochondria Suggests Functional Phosphate-Activated Glutaminase Activity in Matrix

One of the forms of phosphate activated glutaminase (PAG) is associated with the inner mitochondrial membrane. It has been debated whether glutamate formed from glutamine in the reaction catalyzed by PAG has direct access to mitochondrial or cytosolic metabolism. In this study, metabolism of [U-¹³C]glutamine (3 mM) or [U-¹³C]glutamate (10 mM) was investigated in isolated rat brain mitochondria. The presence of a functional tricarboxylic (TCA) cycle in the mitochondria was tested using [U-¹³C]succinate as substrate and extensive labeling in aspartate was seen. Accumulation of glutamine into the mitochondrial matrix was inhibited by histidine (15 mM). Extracts of mitochondria were analyzed for labeling in glutamine, glutamate and aspartate using liquid chromatography-mass spectrometry. Formation of [U-¹³C]glutamate from exogenous [U-¹³C]glutamine was decreased about 50% (P < 0.001) in the presence of histidine. In addition, the ¹³C-labeled skeleton of [U-¹³C]glutamine was metabolized more vividly in the tricarboxylic acid (TCA) cycle than that from [U-¹³C]glutamate, even though glutamate was labeled to a higher extent in the latter condition. Collectively the results show that transport of glutamine into the mitochondrial matrix may be a prerequisite for deamidation by PAG. Special issue article in honor of Dr. Frode Fonnum. Lasse K. Bak and Elżbieta Ziemińska contributed equally to the experimental work described in this paper.     

Complex Glutamate Labeling from [U-13C]glucose or [U-13C]lactate in Co-cultures of Cerebellar Neurons and Astrocytes

Glutamate metabolism was studied in co-cultures of mouse cerebellar neurons (predominantly glutamatergic) and astrocytes. One set of cultures was superfused (90 min) in the presence of either [U-¹³C]glucose (2.5 mM) and lactate (1 mM) or [U-¹³C]lactate (1 mM) and glucose (2.5 mM). Other sets of cultures were incubated in medium containing [U-¹³C]lactate (1 mM) and glucose (2.5 mM) for 4 h. Regardless of the experimental conditions cell extracts were analyzed using mass spectrometry and nuclear magnetic resonance spectroscopy. ¹³C labeling of glutamate was much higher than that of glutamine under all experimental conditions indicating that acetyl-CoA from both lactate and glucose was preferentially metabolized in the neurons. Aspartate labeling was similar to that of glutamate, especially when [U-¹³C]glucose was the substrate. Labeling of glutamate, aspartate and glutamine was lower in the cells incubated with [U-¹³C]lactate. The first part of the pyruvate recycling pathway, pyruvate formation, was detected in singlet and doublet labeling of alanine under all experimental conditions. However, full recycling, detectable in singlet labeling of glutamate in the C-4 position was only quantifiable in the superfused cells both from [U-¹³C]glucose and [U-¹³C]lactate. Lactate and alanine were mostly uniformly labeled and labeling of alanine was the same regardless of the labeled substrate present and higher than that of lactate when superfused in the presence of [U-¹³C]glucose. These results show that metabolism of pyruvate, the precursor for lactate, alanine and acetyl-CoA is highly compartmentalized. Special issue dedicated to John P. Blass.     

Oxidation of Glucose,Ribose, Alanine,and Glutamate by Leishmania braziliensis panamensis1

The metabolism of [1-¹⁴C]- and [6-¹⁴C]glucose, [1-¹⁴]ribose, [1-¹⁴C]- and [U-¹⁴C]alanine, and [1-¹⁴C]- and [5-¹⁴C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of ¹⁴CO₂ produced from [1-¹⁴C]glucose to that from [6-¹⁴C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of ¹⁴CO₂ from [1-¹⁴C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of ¹⁴CO₂ production from [1-¹⁴C]glucose was almost linear with time of incubation, whereas that of [6-¹⁴C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26°C to 34°C had no appreciable effect on the rates or time courses of oxidation of either [1-¹⁴C]- or [6-¹⁴C]glucose or of [1-¹⁴C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of ¹⁴CO₂ produced from [1-¹⁴C]- to [U-¹⁴C]alanine and from [1-¹⁴C]- to [5^-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide. Heating the cultures for 6 or 12 h at 34°C, which converts the promastigotes into an ellipsoidally shaped intermediate form, decreased the rates of oxidation of glucose, alanine, and glutamate. The oxidation of glutamate decreased by about 50% and 70% after a 6-h or 12-h heat treatment, respectively. Returning the heated cultures to 26°C initiated a reversion to the promastigote form and recovery of the rate of glucose oxidation, but glutamate oxidation did not return to control levels by 19 h at 26°C.     

Induced pluripotent stem cells can utilize lactate as a metabolic substrate to support proliferation

Human-induced pluripotent stem cells (iPSCs) hold the promise to improve cell-based therapies. Yet, to meet rising demands and become clinically impactful, sufficient high-quality iPSCs in quantity must be generated, a task that exceeds current capabilities. In this study, K3 iPSCs cultures were examined using parallel-labeling metabolic flux analysis (¹³C-MFA) to quantify intracellular fluxes at relevant bioprocessing stages: glucose concentrations representative of initial media concentrations and high lactate concentrations representative of fed-batch culture conditions, prior to and after bolus glucose feeds. The glucose and lactate concentrations are also representative of concentrations that might be encountered at different locations within 3D cell aggregates. Furthermore, a novel method was developed to allow the isotopic tracer [U-¹³C₃] lactate to be used in the ¹³C-MFA model. The results indicated that high extracellular lactate concentrations decreased glucose consumption and lactate production, while glucose concentrations alone did not affect rates of aerobic glycolysis. Moreover, for the high lactate cultures, lactate was used as a metabolic substrate to support oxidative mitochondrial metabolism. These results demonstrate that iPSCs have metabolic flexibility and possess the capacity to metabolize lactate to support exponential growth, and that high lactate concentrations alone do not adversely impact iPSC proliferation.