CoA is bound to a sulfhydryl group -SH and diffuses away to eventually combine with another acetyl group. This step is irreversible because it is highly exergonic. The rate of this reaction is controlled by negative feedback and the amount of ATP available. If ATP levels increase, the rate of this reaction decreases. If ATP is in short supply, the rate increases. Citrate loses one water molecule and gains another as citrate is converted into its isomer, isocitrate.
Steps 3 and 4. CoA binds the succinyl group to form succinyl CoA. Step 5. A phosphate group is substituted for coenzyme A, and a high- energy bond is formed. This energy is used in substrate-level phosphorylation during the conversion of the succinyl group to succinate to form either guanine triphosphate GTP or ATP. There are two forms of the enzyme, called isoenzymes, for this step, depending upon the type of animal tissue in which they are found. One form is found in tissues that use large amounts of ATP, such as heart and skeletal muscle.
This form produces ATP. The second form of the enzyme is found in tissues that have a high number of anabolic pathways, such as liver. This form produces GTP. In particular, protein synthesis primarily uses GTP. Step 6. Step six is a dehydration process that converts succinate into fumarate. Unlike NADH, this carrier remains attached to the enzyme and transfers the electrons to the electron transport chain directly. Studies performed in recent years have indicated that citrate is not only a powerful sensor and regulator of metabolic pathways but also plays other important roles in different biological processes.
This review will focus on newly discovered features of the physiological and pathological role of citrate inside the cell and its potential use as a therapeutic drug and biomarker. Beyond the well-known role as a metabolic sensor, citrate is involved in the following biological processes: inflammation, cancer, insulin secretion, and histone acetylation.
Two approaches have been used to identify these new functions. The first is based on the inhibition of mitochondrial CIC activity by the specific substrate analogue 1,2,3-benzene-tricarboxylate BTA Joseph et al. Furthermore, direct measurements by liquid chromatography-tandem mass spectrometry van de Wier et al. Finally, the use of citrate as a therapeutic drug was also tentatively tested Zhang et al.
During inflammation, cells undergo significant metabolic changes mainly consisting of a downregulation of bioenergetic pathways.
A genome-wide expression analysis performed on human leukocytes, from subjects treated with bacterial endotoxin LPS , identified a significant decrease in the expression of genes involved in mitochondrial energy metabolism such as genes encoding respiratory chain complex subunits, adenine nucleotide carrier, and PDH and protein synthesis machinery such as the elongation initiation factor complex Calvano et al.
A downregulation of mitochondrial bioenergetic processes has also been observed in LPS-activated macrophages. These cells rapidly switch from resting-state metabolism, which uses oxidative phosphorylation to generate ATP, to an activated state in which glycolysis is greatly induced. Upregulated glycolysis is not only used to generate more ATP per time unit but also to generate other intermediates from the pentose phosphate pathway.
In fact, macrophages show a high requirement for such intermediates to produce a whole range of inflammatory mediators. Mitochondrial enzymes involved in the Krebs cycle are also inhibited, indicating a shift of the TCA cycle from being a purely catabolic pathway generating ATP to being, at least in part, an anabolic pathway.
Acetyl-CoA provides units to synthesize lipids, including arachidonic acid, which is needed for the production of prostaglandin, an intermediate of inflammation.
The cytosolic supply of citrate is provided by the mitochondrial carrier CIC , which plays a regulatory role in the production of inflammation intermediates. Experimental evidence for this regulatory role comes from the specific ablation of CIC using siRNA and CIC activity inhibition, thus limiting the export of citrate from mitochondria and greatly decreasing the production of proinflammatory prostaglandins, ROS and NO.
These findings indicate a key role of mitochondria-derived citrate in LPS induction Infantino et al. Furthermore, the early gene activation of ACLY in LPS as well as in cytokine-induced macrophages suggests a primary role for cytosolic citrate, a substrate of ACLY, which could be a signal molecule in inflammation Infantino et al. The emerging role of citrate in immune cells is part of a growing body of literature that focuses on the interplay among immunity, inflammation, and metabolic changes.
In LPS-activated macrophages, although there is an overall decrease in TCA cycle activity, there is a marked increase in the TCA cycle intermediate succinate, in addition to citrate. LPS also leads to an increase in protein succinylation, although the consequence of these post-translational modification proteins is not yet understood. The meanings of these metabolic changes are not fully understood. The metabolites are not simply consequences of catabolism or anabolism, but they act as specific cell signals.
Thus, by studying metabolic alterations in immune cells, we can better understand the pathogenesis of inflammatory diseases and consider novel treatments.
In cancer cells, a reprogramming of cellular metabolism toward macromolecules synthesis is critical to supply enough nucleotides, proteins, and lipids to proliferate and build new cells. Thus, glycolysis together with the pentose phosphate pathway becomes the best way to synthesize some metabolic intermediates for biosynthesis, such as ribose, glycerol, serine, NADPH, etc. Recently, Catalina-Rodriguez et al. As a consequence of CIC inhibition, the tumor cells die.
Therefore, it is conceivable that CIC inhibitors could provide a platform for the design of a new class of anticancer drugs. Citrate-derived acetyl-CoA is also used in the mevalonate pathway leading to prenylation, a process required for the ability of Ras and Rho proteins to induce malignant transformation invasion and metastasis Armstrong et al.
Increased production of acetyl-CoA does not promote ketogenesis. Myc also promotes mitochondrial biogenesis and nucleotide and amino acid synthesis. Glutamine has an important role in cancer metabolism Figure 3. It is a carbon unit supplier in proliferating cancer cells because it provides both OAA and acetyl-CoA for citrate production Reitzer et al.
The transcriptional factor Myc increases glutamine uptake and its conversion into glutamate by promoting expression of glutaminase. Glutamine also provides amine groups for other biosynthetic processes such as pyrimidine and purine synthesis.
HIF1 stimulates the expression of many target glycolytic enzymes and blocks the use of pyruvate by mitochondrial PDH. By diverting pyruvate into lactate, the so-called aerobic glycolysis or Warburg effect Warburg, ; Israel and Schwartz, , HIF1 blocks carbon incorporation into mitochondrial citrate, which is critical for lipid biosynthesis. Therefore, the HIF1 conditions may exert some anti-proliferative effect as observed in hematopoietic and renal cells Lum et al.
When glucose is diverted from mitochondrial acetyl-CoA and citrate production, proliferating cancer cells may use a reductive and carboxylating biosynthetic reaction from glutamine instead of the oxidative metabolism of both glucose and glutamine Le et al. The citrate exported to the cytosol may in part be metabolized in the oxidative direction by isocitrate dehydrogenase 1 IDH1 and contribute to a shuttle that produces cytosolic NADPH Ward and Thompson, Figure 2.
This shuttle promotes electron transport from mitochondria when the activity of electron transport chain is inhibited as in hypoxic conditions. Citrate synthesis via reductive carboxylation.
Citrate is exported to the cytosol, where it is in part used for lipogenesis, and some may be metabolized in the oxidative direction by IDH1 and contribute to a shuttle that produces cytosolic NADPH. Although it still remains unclear if 2-HG is truly a pathogenic oncometabolite resulting from IDH mutations or if it is just the byproduct of a loss-of-function mutation, its measurement in primary tumors with unknown IDH mutation status could serve as a biomarker for both IDH1 and IDH2 mutations Ward and Thompson, Concerning the biological role of 2-HG, some hypothesis have been proposed.
Evidence from myeloid cells overexpressing IDH mutants Figueroa et al. This results in altered histone methylation marks and dysregulated cellular differentiation.
Taking into consideration that the CIC gene has previously been implicated in epigenetic and cancer biology Morciano et al. It is interesting to note that 2-HG can also increase in the absence of metabolic enzyme mutations. The ability of 2-HG to alter epigenetics may reflect its evolutionary ancient status as signal for elevated oxygen deficiency. Finally, beyond the 2-HG involvement as oncometabolite, others Krebs cycle-related enzymes have previously been identified as oncometabolite.
Mutations in SDH and fumarate hydratase FH give rise to increase of succinate and fumarate levels in renal carcinoma, pheochromocytoma, and paraganglioma Isaacs et al. However, the role of these metabolites in tumorigenesis is not clear. It is not excluded an involvement of alteration of histone demethylase activity, as found by Smith et al. However, these findings highlight an increasing involvement of TCA cycle enzymes and metabolites in tumor formation.
This cycle involves the exit of citrate and isocitrate from the mitochondria via CIC. In the cytosol, citrate can be converted to isocitrate by cytosolic aconitase. From studies performed on abnormal insulin secretion induced by the antipsychotic drug clozapine, we have further demonstrated the involvement of citrate as a signaling molecule and CIC in insulin secretion through its transcriptional regulator FOXA1 Menga et al. Incubation of spermatic cells with a high glucose concentration Interestingly, CIC inhibition reduced sperm hyperactivated motility and acrosome reaction.
Moreover, incubation of sperm cells with citrate induced insulin secretion and triggered the activity of these sperms. Chromatin remodeling through histone modification greatly affects the accessibility of DNA and regulates specific gene expression Sterner and Berger, ; Kurdistani and Grunstein, ; Li et al.
Among the modifications, acetylation and deacetylation processes, catalyzed by classes of histone acetyltransferases and histone deacetylases, respectively, are the most common modifications. The acetylation process is dynamically regulated by physiological changes in concentration of acetyl-CoA derived from the citrate exported by CIC outside the mitochondria and produced by ATP citrate lyase.
Experimental evidence indicates a significant link between cellular metabolism and histone acetylation. Wellen et al. However, the acetylation of histones does not depend on ACLY activity but is linked to the cytosolic citrate pool, suggesting that a crucial role is performed by the CIC as a citrate supplier.
Morciano et al. Different defects of early brain development have been associated with defects of mitochondrial respiratory chain and Krebs cycle, including PDH deficiency, SLCA25A19 defect, aconitase deficiency, fumarate deficiency, and complex I assembly defect Rosenberg et al. Recently, agenesis of the corpus callosum, a birth defect that occurs in different human congenital syndromes, and optic nerve hypoplasia have been associated with mutations in the SLC25A1 gene MIM Edvardson et al.
Two mutations GD and RH , located in highly conserved positions of CIC through evolution, significantly affect protein function, as demonstrated by experiments performed with a yeast strain harboring human CIC mutations at equivalent positions in the orthologous yeast protein, which exhibits a growth defect under stress condition and a marked loss of citrate transport activity in reconstituted liposomes Edvardson et al.
Increased 2-HG is most likely caused by the inability of the mitochondria to export citrate and isocitrate into the cytosol, giving rise to increased mitochondrial concentration and subsequent higher excretion of intermediates downstream isocitrate.
In relation to the latter defect, a recent study from Nota et al. Based on these findings, in addition to detection of mutations in the d HG dehydrogenase gene and isocitrate dehydrogenase, detection of CIC gene mutations should be included in the analysis of patients with neurometabolic diseases, such hydroxyglutaric aciduria.
All patients with CIC gene defects showed an impaired mitochondria citrate efflux, demonstrated by stable isotope labeling experiments and the absence or marked reduction of CIC activity in fibroblasts.
This new finding provides new basis in understanding the pathophysiology of this disease. This effect is a consequence of high levels of free fatty acids and glucose associated with metabolic syndrome. In combination with hydrogen peroxide, elevated citrate levels promote oxidative stress, whereas citrate alone has no effect on oxidative stress, suggesting that citrate acts only indirectly van de Wier et al.
The molecular mechanism of this interaction is not clear. It is likely that citrate stimulates hydroxyl radical formation from hydrogen peroxide in the presence of iron through the formation of an iron-citrate complex Gutteridge, Because iron is stored as ferritin, it has to be released to promote radical formation.
Release requires chelation of iron with citrate or other chelating agents , suggesting that citrate could be an iron recruiter rather than a direct radical inducer Goddard et al. Because citrate is an essential intermediate located at the crossroad of metabolism and a key regulator of energy production, its use in cancer treatment has been hypothesized and tentatively tested. An excess of citrate in cancer cells would inhibit all PFK isoforms, leading to an arrest of glycolysis, inhibition of all ATP production pathways, and stimulation of ATP-consuming pathways.
This metabolic condition would result in ATP depletion, leading to arrest of cancer cell growth and cell death Zhang et al. MSTOH mesothelioma cells continuously exposed to citrate show a down-proliferation or cell death, depending on the citrate concentration. When citrate is removed from the medium, culture cancer cells restart to grow.
Interestingly, this regrowth is hampered in presence of the drug cisplatin, suggesting a synergistic effect between citrate and cisplatin Zhang et al. The mechanism of this effect is not completely known. Moreover, citrate could also sensitize cells to cisplatin through the inhibition of the anti-apoptotic protein Mc and Bcl-x L , which are overexpressed in mesothelioma cells Willis et al.
Recently, a lethal effect of citrate in different cell lines Tet21N, Sk-N-SH, and U , through the activation of caspases 8 and 2, has been demonstrated Kruspig et al. Among all mammalian organs, the prostate possesses the particular feature of accumulating high amounts of citrate, reaching up to m m in the prostatic fluid Kavanagh, ; Mazurek et al. Therefore, the intermediary metabolism of prostatic cell is modified by their specialized function of net citrate production.
Whereas two of the six carbons from citrate are stored as OAA and two are lost as CO 2 in the normal mammalian metabolism, all six carbons are removed from the metabolic pools as an end product of metabolism in the citrate-producing prostatic cells. However, to maintain this high cellular level, citrate oxidation must be inhibited.
Learning Objectives Describe the fate of the acetyl CoA carbons in the citric acid cycle. Acetyl CoA transfers its acetyl group to oxaloacetate to form citrate and begin the citric acid cycle. This reaction requires that the cell use up some energy by breaking down an ATP molecule. Fatty acids are made by repeatedly joining together the two-carbon fragments found in acetyl-CoA and then reducing the -CO- part of the molecule to -CH 2 -.
In this way, the hydrocarbon chain, which will become the hydrophobic, energy storing part of the fatty acid, grows two-carbons at a time as the cycle of joining reactions is repeated over and over again.
Most of these reactions take place in and on the membranes of the endoplasmic reticulum known as microsomal membranes and takes place in several stages. In step three, an acetyl-group is attached to the cysteine-SH carrier site on the FAS complex and a malonyl-group is attached to the pantethine-SH carrier site.
In a joining reaction, the acetyl-group is transfered to and joined to the malonyl-group with the simultaneous expulsion of a CO 2 molecule. This is the elongation reaction called a condensation reaction and the hydrocarbon part of the new fatty acid is now four-carbons long. When the hydrocarbon chain of the new fatty acid is 16 carbon atoms long the bond joining the fatty acid to the pantetheine-SH carrier site is finally broken and the C saturated fatty acid palmitate is released.
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