Kinetic characterization of vero cell metabolism in a serum‐free batch culture process

A global kinetic study of the central metabolism of Vero cells cultivated in a serum‐free medium is proposed in the present work. Central metabolism including glycolysis, glutaminolysis, and tricarboxylic acid cycle (TCA) was demonstrated to be saturated by high flow rates of consumption of the two major substrates, glucose, and glutamine. Saturation was reavealed by an accumulation of metabolic intermediates and amino acids, by a high production of lactate needed to balance the redox pathway, and by a low participation of the carbon flow to the TCA cycle supply. Different culture conditions were set up to reduce the central metabolism saturation and to better balance the metabolic flow rates between lactate production and energetic pathways. From these culture conditions, substitutions of glutamine by other carbon sources, which have lower transport rates such as asparagine, or pyruvate in order to shunt the glycolysis pathway, were successful to better balance the central metabolism. As a result, an increase of the cell growth with a concomitant decrease of cell death and a better distribution of the carbon flow between TCA cycle and lactate production occurred. We also demonstrated that glutamine was a major carbon source to supply the TCA cycle in Vero cells and that a reduction of lactate production did not necessary improve the efficiency of the Vero cell metabolism. Thus, to adapt the formulation of the medium to the Vero cell needs, it is important to provide carbon substrates inducing a regulated supply of carbon in the TCA cycle either through the glycolysis or through other pathways such as glutaminolysis. Finally, this study allowed to better understand the Vero cell behavior in serum‐free medium which is a valuable help for the implementation of this cell line in serum‐free industrial production processes. Biotechnol. Bioeng. 2010;107: 143–153. © 2010 Wiley Periodicals, Inc.     

Reverse TCA cycle flux through isocitrate dehydrogenases 1 and 2 is required for lipogenesis in hypoxic melanoma cells

The tricarboxylic acid (TCA) cycle is the central hub of oxidative metabolism, running in the classic forward direction to provide carbon for biosynthesis and reducing agents for generation of ATP. Our metabolic tracer studies in melanoma cells showed that in hypoxic conditions the TCA cycle is largely disconnected from glycolysis. By studying the TCA branch point metabolites, acetyl CoA and citrate, as well as the metabolic endpoint glutamine and fatty acids, we developed a comprehensive picture of the rewiring of the TCA cycle that occurs in hypoxia. Hypoxic tumor cells maintain proliferation by running the TCA cycle in reverse. The source of carbon for acetyl CoA, citrate, and fatty acids switches from glucose in normoxia to glutamine in hypoxia. This hypoxic flux from glutamine into fatty acids is mediated by reductive carboxylation. This reductive carboxylation is catalyzed by two isocitrate dehydrogenases, IDH1 and IDH2. Their combined action is necessary and sufficient to effect the reverse TCA flux and maintain cellular viability.     

Glutamine fuels proliferation but not migration of endothelial cells

Endothelial metabolism is a key regulator of angiogenesis. Glutamine metabolism in endothelial cells (ECs) has been poorly studied. We used genetic modifications and ¹³C tracing approaches to define glutamine metabolism in these cells. Glutamine supplies the majority of carbons in the tricyclic acid (TCA) cycle of ECs and contributes to lipid biosynthesis via reductive carboxylation. EC‐specific deletion in mice of glutaminase, the initial enzyme in glutamine catabolism, markedly blunts angiogenesis. In cell culture, glutamine deprivation or inhibition of glutaminase prevents EC proliferation, but does not prevent cell migration, which relies instead on aerobic glycolysis. Without glutamine catabolism, there is near complete loss of TCA intermediates, with no compensation from glucose‐derived anaplerosis. Mechanistically, addition of exogenous alpha‐ketoglutarate replenishes TCA intermediates and rescues cellular growth, but simultaneously unveils a requirement for Rac1‐dependent macropinocytosis to provide non‐essential amino acids, including asparagine. Together, these data outline the dependence of ECs on glutamine for cataplerotic processes; the need for glutamine as a nitrogen source for generation of biomass; and the distinct roles of glucose and glutamine in EC biology.     

NMR Spectroscopy of Cultured Astrocytes: Effects of Glutamine and the Gliotoxin Fluorocitrate

Abstract: Glial synthesis of glutamine, citrate, and other carbon skeletons, as well as metabolic effects of the gliotoxin fluorocitrate, were studied in cultured astrocytes with ¹³C and ³¹P NMR spectroscopy. f2–¹³C]Acetate and [1–¹³C]glucose were used as labeled precursors. In some experiments glutamine (2.5 mM) was added to the culture medium. Fluorocitrate (20 μM) inhibited the tricarboxylic acid (TCA) cycle without affecting the level of ATP. The net export of glutamine was reduced significantly, and that of citrate increased similarly, consistent with an inhibition of aconitase. Fluorocitrate (100 μM) inhibited TCA cycle activity even more and (without addition of glutamine) caused a 40% reduction in the level of ATP. In the presence of 2.5 mM glutamine, 100 μM fluorocitrate did not affect ATP levels, although glutamine synthesis was nearly fully blocked. The consumption of the added glutamine increased with increasing concentrations of fluorocitrate, whereas the consumption of glucose decreased. This shows that glutamine fed into the TCA cycle, substituting for glucose as an energy substrate. These findings may explain how fluorocitrate selectively lowers the level of glutamine and inhibits glutamine formation in the brain in vivo, viz., not by depleting glial cells of ATP, but by causing a rerouting of 2-oxoglutarate from glutamine synthesis into the TCA cycle during inhibition of aconitase. Analysis ^; of the ¹³C labeling of the C-2 versus the C-4 positions in glutamine obtained with [2–¹³C]acetate revealed that 57% of the TCA cycle intermediates were lost per turn of the cycle. Glutamine and citrate were the main TCA cycle intermediates to be released, but a large amount of lactate formed from TCA cycle intermediates was also released, showing that recycling of pyruvate takes place in astrocytes.     

Pyruvate decarboxylation in astrocytes and in neurons in primary cultures in the presence and the absence of ammonia

Oxidative decarboxylation of [1-¹⁴C]pyruvate was studied in primary cultures of neurons and of astrocytes. The rate of this process, which is a measure of carbon flow into the tricarboxylic acid (TCA) cycle and which is inhibited by its end product, acetyl CoA, was determined under conditions which would either elevate or reduce the components of the malate-aspartate shuttle (MAS). Addition of aspartate (1 mM) was found to stimulate pyruvate decarboxylation in astrocytes whereas addition of glutamate (or glutamine) had no effect. Since aspartate is a precursor for extramitochondrial malate, and thus intramitochondrial oxaloacetate, whereas glutamate and glutamine are not, this suggests that an increase in oxaloacetate level stimulates TCA cycle activity. Conversely, a reduction of the glutamate content by 3 mM ammonia, which might reduce exchange between glutamate and aspartate across the mitochondrial membrane, suppressed pyruvate decarboxylation. This effect was abolished by addition of glutamate or glutamine or exposure to methionine sulfoximine (MSO). These findings suggest that impairment of MAS activity by removal of MAS constituents decreases TCA cycle activity whereas replenishment of these compounds restores the activity of the TCA cycle. No corresponding effects were observed in neurons.     

Viral effects on metabolism: changes in glucose and glutamine utilization during human cytomegalovirus infection

Human cytomegalovirus (HCMV) infection causes dramatic alterations of intermediary metabolism, similar to those found in tumor cells. In infected cells, glucose carbon is not completely broken down by the tricarboxylic acid (TCA) cycle for energy; instead, it is used biosynthetically. This process requires increased glucose uptake, increased glycolysis and the diversion of glucose carbon, in the form of citrate, from the TCA cycle for use in HCMV-induced fatty acid biosynthesis. The diversion of citrate from the TCA cycle (cataplerosis) requires induction of enzymes to promote glutaminolysis, the conversion of glutamine to α-ketoglutarate to maintain the TCA cycle (anaplerosis) and ATP production. Such changes could result in heretofore uncharacterized pathogenesis, potentially implicating HCMV as a subtle cofactor in many maladies, including oncogenesis. Recognition of the effects of HCMV, and other viruses, on host cell metabolism will provide new understanding of viral pathogenesis and novel avenues for antiviral therapy.     

Metabolic shifts toward glutamine regulate tumor growth,invasion and bioenergetics in ovarian cancer

Glutamine can play a critical role in cellular growth in multiple cancers. Glutamine‐addicted cancer cells are dependent on glutamine for viability, and their metabolism is reprogrammed for glutamine utilization through the tricarboxylic acid (TCA) cycle. Here, we have uncovered a missing link between cancer invasiveness and glutamine dependence. Using isotope tracer and bioenergetic analysis, we found that low‐invasive ovarian cancer (OVCA) cells are glutamine independent, whereas high‐invasive OVCA cells are markedly glutamine dependent. Consistent with our findings, OVCA patients’ microarray data suggest that glutaminolysis correlates with poor survival. Notably, the ratio of gene expression associated with glutamine anabolism versus catabolism has emerged as a novel biomarker for patient prognosis. Significantly, we found that glutamine regulates the activation of STAT3, a mediator of signaling pathways which regulates cancer hallmarks in invasive OVCA cells. Our findings suggest that a combined approach of targeting high‐invasive OVCA cells by blocking glutamine's entry into the TCA cycle, along with targeting low‐invasive OVCA cells by inhibiting glutamine synthesis and STAT3 may lead to potential therapeutic approaches for treating OVCAs.     

The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and -ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the -ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the -ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-¹³C]glucose (2.5 mM) and isoleucine (1 mM) or [U-¹³C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of ¹³C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-¹³C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-¹³C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-¹³C]glucose. Labeling in glutamate and aspartate from [U-¹³C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U-¹³C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.     

Glucose alleviates ammonia-induced inhibition of short-chain fatty acid metabolism in rat colonic epithelial cells

Ammonia decreased metabolism by rat colonic epithelial cells of butyrate and acetate to CO2 and ketones but increased oxidation of glucose and glutamine. Ammonia decreased cellular concentrations of oxaloacetate for all substrates evaluated. The extent to which butyrate carbon was oxidized to CO2 after entering the tricarboxylic acid (TCA) cycle was not significantly influenced by ammonia, suggesting there was no major shift toward efflux of carbon from the TCA cycle. Ammonia reduced entry of butyrate carbon into the TCA cycle, and the proportion of CoA esterified with acetate and butyrate correlated positively with the production of CO2 and ketone bodies. Also, ammonia reduced oxidation of propionate but had no effect on oxidation of 3-hydroxybutyrate. Inclusion of glucose, lactate, or glutamine with butyrate and acetate counteracted the ability of ammonia to decrease their oxidation. In rat colonocytes, it appears that ammonia suppresses short-chain fatty acid (SCFA) oxidation by inhibiting a step before or during their activation. This inhibition is alleviated by glucose and other energy-generating compounds. These results suggest that ammonia may only affect SCFA metabolism in vivo when glucose availability is compromised.     

Insights into the central metabolism of Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5) insect cells by radiolabeling studies

The insect cell baculovirus expression vector system (BEVS) is one of the most commonly used expression systems for recombinant protein production. This system is also widely used for the production of recombinant virus and virus-like particles. Although several published reports exist on recombinant protein expression using insect cells, information dealing with their metabolism in vitro is relatively scarce. In this work we have analyzed the metabolism of glucose and glutamine, the main carbon and/or energy compounds, of the two most commonly used insect cell lines, Spodoptera frugiperda (Sf-9) and the Trichoplusia ni BTI-Tn-5B1-4 (Tn-5). Radiolabeled substrates have been used to determine the flux of glucose carbon entering the tricarboxylic acid cycle (TCA) and the pentose phosphate (PP) pathway by direct measurement of 14CO2 produced. The percentage of total glucose metabolized to CO2 via the TCA cycle was higher in the case of the Sf-9 (2.7%) compared to Tn-5 (0.6%) cells, while the percentage of glucose that is metabolized via the PP pathway was comparable at 14% and 16% for the two cell lines, respectively. For both cell lines, the remaining 83% of glucose is metabolized through other pathways generating, for example, lactate, alanine, etc. The percentage of glutamine oxidized in the TCA cycle was approximately 5-fold higher in the case of the Tn-5 (26.1%) as compared to the Sf-9 cells (4.6%). Furthermore, the changes in the metabolic fluxes of glucose and glutamine in Tn-5-PYC cells, which have been engineered to express a cytosolic pyruvate carboxylase, have been studied and compared to the unmodified cells Tn-5. As a result of this metabolic engineering, significant increase in the percentage of glucose oxidized in the TCA cycle (3.2%) as well as in the flux through the PP pathway (34%) of the Tn-5-PYC were observed.     

Role of glutamine and interlinked asparagine metabolism in vessel formation

Endothelial cell (EC) metabolism is emerging as a regulator of angiogenesis, but the precise role of glutamine metabolism in ECs is unknown. Here, we show that depriving ECs of glutamine or inhibiting glutaminase 1 (GLS1) caused vessel sprouting defects due to impaired proliferation and migration, and reduced pathological ocular angiogenesis. Inhibition of glutamine metabolism in ECs did not cause energy distress, but impaired tricarboxylic acid (TCA) cycle anaplerosis, macromolecule production, and redox homeostasis. Only the combination of TCA cycle replenishment plus asparagine supplementation restored the metabolic aberrations and proliferation defect caused by glutamine deprivation. Mechanistically, glutamine provided nitrogen for asparagine synthesis to sustain cellular homeostasis. While ECs can take up asparagine, silencing asparagine synthetase (ASNS, which converts glutamine‐derived nitrogen and aspartate to asparagine) impaired EC sprouting even in the presence of glutamine and asparagine. Asparagine further proved crucial in glutamine‐deprived ECs to restore protein synthesis, suppress ER stress, and reactivate mTOR signaling. These findings reveal a novel link between endothelial glutamine and asparagine metabolism in vessel sprouting.     

Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate.

The sucA gene, encoding the E1 component of alpha-ketoglutarate dehydrogenase, was cloned from Bradyrhizobium japonicum USDA110, and its nucleotide sequence was determined. The gene shows a codon usage bias typical of non-nif and non-fix genes from this bacterium, with 89.1% of the codons being G or C in the third position. A mutant strain of B. japonicum, LSG184, was constructed with the sucA gene interrupted by a kanamycin resistance marker. LSG184 is devoid of alpha-ketoglutarate dehydrogenase activity, indicating that there is only one copy of sucA in B. japonicum and that it is completely inactivated in the mutant. Batch culture experiments on minimal medium revealed that LSG184 grows well on a variety of carbon substrates, including arabinose, malate, succinate, beta-hydroxybutyrate, glycerol, formate, and galactose. The sucA mutant is not a succinate auxotroph but has a reduced ability to use glutamate as a carbon or nitrogen source and an increased sensitivity to growth inhibition by acetate, relative to the parental strain. Because LSG184 grows well on malate or succinate as its sole carbon source, we conclude that B. japonicum, unlike most other bacteria, does not require an intact tricarboxylic acid (TCA) cycle to meet its energy needs when growing on the four-carbon TCA cycle intermediates. Our data support the idea that B. japonicum has alternate energy-yielding pathways that could potentially compensate for inhibition of alpha-ketoglutarate dehydrogenase during symbiotic nitrogen fixation under oxygen-limiting conditions.     

Glutamate synthesis has to be matched by its degradation – where do all the carbons go?

The central process in energy production is the oxidation of acetyl‐CoA to CO₂ by the tricarboxylic acid (TCA, Krebs, citric acid) cycle. However, this cycle functions also as a biosynthetic pathway from which intermediates leave to be converted primarily to glutamate, GABA, glutamine and aspartate and to a smaller extent to glucose derivatives and fatty acids in the brain. When TCA cycle ketoacids are removed, they must be replaced to permit the continued function of this essential pathway, by a process termed anaplerosis. Since the TCA cycle cannot act as a carbon sink, anaplerosis must be coupled with cataplerosis; the exit of intermediates from the TCA cycle. The role of anaplerotic reactions for cellular metabolism in the brain has been studied extensively. However, the coupling of this process with cataplerosis and the roles that both pathways play in the regulation of amino acid, glucose, and fatty acid homeostasis have not been emphasized. The concept of a linkage between anaplerosis and cataplerosis should be underscored, because the balance between these two processes is essential. The hypothesis that cataplerosis in the brain is achieved by exporting the lactate generated from the TCA cycle intermediates into the blood and perivascular area is presented. This shifts the generally accepted paradigm of lactate generation as simply derived from glycolysis to that of oxidation and might present an alternative explanation for aerobic glycolysis.

     

Comparative metabolic flux profiling of melanoma cell lines: beyond the Warburg effect

Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the "reverse" (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma.     

Regulation of carbon and nitrogen utilization by CbrAB and NtrBC two-component systems in Pseudomonas aeruginosa

The global effect of the CbrAB and NtrBC two-component systems on the control of carbon and nitrogen utilization in Pseudomonas aeruginosa was characterized by phenotype microarray analyses with single and double mutants and the isogenic parent strain. The tested compounds were clustered based on the growth phenotypes of these strains, and the results clearly demonstrated the pivotal roles of CbrAB and NtrBC in carbon and nitrogen utilization, respectively. Growth of the cbrAB deletion mutant on arginine, histidine, and polyamines used as the sole carbon source was abolished, while growth on the tricarboxylic acid (TCA) cycle intermediates was sustained. In this study, suppressors of the cbr mutant were selected from minimal medium containing l-arginine as the sole carbon and nitrogen source. These mutants fell into two groups according to the ability to utilize histidine. The genomic library of a histidine-positive suppressor mutant was constructed, and the corresponding suppressor gene was identified by complementation as an ntrB allele. Similar results were obtained from four additional suppressor mutants, and point mutations of these ntrB alleles resulting in the following changes in residues were identified, with implications for reduced phosphatase activities: L126W, D227A, P228L, and S229I. The Ntr systems of these ntrB mutants became constitutively active, as revealed by the activity profiles of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase. As a result, these mutants not only regain the substrate-specific induction on catabolic arginine and histidine operons but are also expressed to higher levels than the wild type. While the DeltacbrAB ntrB(Con) mutant restored growth on many N-containing compounds used as the carbon sources, its capability to grow on TCA cycle intermediates and glucose was compromised when ammonium served as the sole nitrogen source, mostly due to an extreme imbalance of carbon and nitrogen regulatory systems. In summary, this study supports the notion that CbrAB and NtrBC form a network to control the C/N balance in P. aeruginosa. Possible molecular mechanisms of these two regulatory elements in the control of arginine and histidine operons used as the model systems are discussed.     

Glutamine Metabolism Is Essential for Human Cytomegalovirus Infection

Human fibroblasts infected with human cytomegalovirus (HCMV) were more viable than uninfected cells during glucose starvation, suggesting that an alternate carbon source was used. We have determined that infected cells require glutamine for ATP production, whereas uninfected cells do not. This suggested that during infection, glutamine is used to fill the tricarboxylic acid (TCA) cycle (anaplerosis). In agreement with this, levels of glutamine uptake and ammonia production increased in infected cells, as did the activities of glutaminase and glutamate dehydrogenase, the enzymes needed to convert glutamine to α-ketoglutarate to enter the TCA cycle. Infected cells starved for glutamine beginning 24 h postinfection failed to produce infectious virions. Both ATP and viral production could be rescued in glutamine-starved cells by the TCA intermediates α-ketoglutarate, oxaloacetate, and pyruvate, confirming that in infected cells, a program allowing glutamine to be used anaplerotically is induced. Thus, HCMV infection activates the mechanisms needed to switch the anaplerotic substrate from glucose to glutamine to accommodate the biosynthetic and energetic needs of the viral infection and to allow glucose to be used biosynthetically.Glucose (Glc) and glutamine are the two most abundant nutrients used by mammalian cells. They are necessary for the generation of energy, macromolecules, and second messengers (1, 5-7, 9-12, 16). Glucose has long been considered absolutely essential for the viability of mammalian cells because of its contribution to energy homeostasis through glycolysis and the tricarboxylic acid (TCA) cycle (Fig. ​(Fig.1).1). Recent studies demonstrated that human diploid fibroblasts are killed by glucose deprivation by a mechanism different from apoptosis (20).Open in a separate windowFIG. 1.Glycolysis and the citric acid cycle showing glucose and glutamine utilization. The aspects of the cytoplasmic (Cyto) and mitochondrial (Mito) metabolism of glucose and glutamine discussed in the text are outlined. Dashed lines indicate that there are several intermediates formed (several reactions) between the ones shown. PEPCK, phosphoenolpyruvate carboxykinase; ME: malic enzyme; GDH, glutamate dehydrogenase; GLS, glutaminase; ACL, ATP citrate lyase; OAA, oxaloacetic acid; AcCoA, acetyl coenzyme A.In 1924, Warburg observed that cancer cells metabolize glucose very differently than normal cells (18). Cancer cells converted glucose into lactate even in the presence of sufficient oxygen to support mitochondrial oxidative phosphorylation (Fig. ​(Fig.1).1). This utilization of glucose, called the Warburg effect, results in only 2 ATP molecules produced per molecule of glucose, whereas if it had proceeded through the TCA cycle and mitochondrial oxidative phosphorylation, an additional 36 ATP molecules would have been produced per molecule of glucose. Recently reported data provide an explanation for what appeared to be an inefficient utilization of glucose (7, 8, 19). In cancer cells, exogenous glutamine is used as a carbon source, which facilitates the cell''s ability to use glucose biosynthetically instead of breaking it down completely for energy. This is accomplished by glutamine being converted to α-ketoglutarate via glutaminase (GLS) and glutamate dehydrogenase (GDH) (Fig. ​(Fig.1).1). This process of replenishing TCA cycle intermediates is called anaplerosis. Thus, glutamine anaplerotically fills the TCA cycle (Fig. ​(Fig.1),1), providing NADH for oxidative phosphorylation as well as TCA cycle intermediates, which serve as important biosynthetic precursors (7, 8). In contrast, normal cells are believed to use only a small amount of consumed glutamine for macromolecular biosynthesis and energy; thus, glucose and glutamine metabolism are dramatically altered in tumor cells (8, 16).While glutamine starvation in many cell types has little impact on cell viability, it has been shown to induce cell death in cancer cell lines that overexpress the oncogene c-myc (20). These cells also showed decreased levels of ATP production correlating with decreased concentrations of TCA cycle intermediates; both are predictable consequences of glutamine starvation if glutamine is being used anaplerotically. In agreement with this finding, the effects of glutamine starvation could be reversed by the addition of the TCA cycle intermediates pyruvate (Pyr) and oxaloacetate (OAA) (Fig. ​(Fig.11).Human cytomegalovirus (HCMV) is a slow-growing betaherpesvirus that exerts a large energetic and biosynthetic demand on cells to ensure successful viral replication. Recent mass spectrometry-based metabolic flux studies indicated global metabolic upregulation in infected cells (14, 15). This included greatly increased glycolysis in which the vast majority of glucose-derived acetyl coenzyme A (AcCoA) went to support fatty acid synthesis (Fig. ​(Fig.1)1) to make membranes needed by the virus. Thus, there is a great decrease in the amount of glucose-derived carbon entering the TCA cycle. In other words, the virus induces a modified Warburg effect so that glucose-derived carbon can be used biosynthetically. These metabolomic data also suggest that glutamine may be used to anaplerotically fill the TCA cycle.We have investigated the impact of glucose and glutamine on HCMV replication. We have found that under conditions of glucose deprivation, infected cells are more viable than mock-infected cells. Thus, we hypothesized that the infected cells use glutamine anaplerotically. In agreement with this prediction, glutamine was found to be necessary for ATP production in infected cells but not in uninfected cells. Furthermore, cells starved of glutamine beginning 24 h postinfection (hpi) failed to produce infectious virions. HCMV-induced glutaminolysis was indicated by increased glutamine uptake and ammonia production corresponding to increased activities of glutaminase and glutamate dehydrogenase. These enzymes convert glutamine to α-ketoglutarate (α-KG) for anaplerotic use in the TCA cycle. The anaplerotic use of glutamine in the TCA cycle was also demonstrated by the finding that both ATP production and viral growth could be rescued by replacing glutamine with the TCA cycle intermediate α-ketoglutarate, oxaloacetate, or pyruvate. Thus, our data suggest that in HCMV-infected cells, as in many tumor cells, a program is activated whereby glutamine utilization increases specifically to maintain the TCA cycle, allowing glucose to be used biosynthetically.     

Comparison of metabolic flux distributions for MDCK cell growth in glutamine- and pyruvate-containing media

In mammalian cell cultures, ammonia that is released into the medium as a result of glutamine metabolism and lactate that is excreted due to incomplete glucose oxidation are both known to essentially inhibit the growth of cells. For some cell lines, for example, hybridoma cells, excreted ammonia also has an effect on product formation. Although glutamine has been generally considered as the major energy source for mammalian cells, it was recently found that various adherent cell lines (MDCK, CHO-K1, and BHK21) can grow as well in glutamine-free medium, provided glutamine is substituted with pyruvate. In such a medium the level of both ammonia and lactate released was significantly reduced. In this study, metabolic flux analysis (MFA) was applied to Madin Darby Canine Kidney (MDCK) cells cultivated in glutamine-containing and glutamine-free medium. The results of the MFA allowed further investigation of the influence of glutamine substitution with pyruvate on the metabolism of MDCK cells during different growth stages of adherent cells, e.g., early exponential and late contact-inhibited phase. Pyruvate seemed to directly enter the TCA cycle, whereas most of the glucose consumed was excreted as lactate. Although the exact mechanisms are not clear so far, this resulted in a reduction of the glucose uptake necessary for cellular metabolism in glutamine-free medium. Furthermore, consumption of ATP by futile cycles seemed to be significantly reduced when substituting glutamine with pyruvate. These findings imply that glutamine-free medium favors a more efficient use of nutrients by cells. However, a number of metabolic fluxes were similar in the two cultivations considered, e.g., most of the amino acid uptake and degradation rates or fluxes through the branch of the TCA cycle converting alpha-ketoglutarate to malate, which is responsible for the mitochondrial ATP synthesis. Besides, the specific rate of cell growth was approximately the same in both cultivations. Thus, the switch from glutamine-containing to glutamine-free medium with pyruvate provided a series of benefits without dramatic changes of cellular metabolism.     

Operation and function of the tricarboxylic acid cycle in the illuminated leaf

Respiratory activity of plants in the light, measured as carbon dioxide release from the tricarboxylic acid (TCA) cycle or oxygen consumption by the respiratory chain, is generally reported to lie between 25 and 100% of that in the dark. While this has been interpreted as evidence for an inhibition of respiration during photosynthesis, an increasing body of evidence indicates that mitochondrial respiration plays an important role in photosynthetic tissues. Historically, the view from experiments using specific respiratory inhibitors has been that oxidative phosphorylation in the mitochondria provides the cytosol with adenosine triphosphate even in the light. However, functioning of TCA cycle reactions is also required for the export of carbon skeletons necessary for nitrate reduction in the cytosol. In addition, export of TCA cycle-derived reducing equivalents may also be necessary for photorespiration (for hydroxypyruvate reduction in the peroxisomes). The work with respiratory inhibitors has recently been complemented by a range of transgenic experiments that provide direct evidence for the importance of the TCA cycle in the illuminated leaves. These transgenesis experiments hint at an important role for ascorbate in coordinating the major pathways of energy metabolism within the leaf and are in keeping with current thinking that redox signals emanating from the mitochondria are important in setting the cellular machinery to maintain overall redox balance. In this review we intend to synthesize recent experimental data to postulate a model of the function of the TCA cycle in the illuminated leaf.