Structural composition of NaCT
The structure of either human or the mouse NaCTs has not been crystallized. The current knowledge for the structural basis of the NaCTs is based upon Vibrio cholerae bacterial dicarboxylate transporter VcINDY. The structure VcINDY greatly differs from the human NaCT but still acts as the best option for the modeling studies [11]. It has a total of 11 transmembrane (TM) helices hairpin loops marked as HPin (hairpin in) and HPout(hairpin out). The TM 4, 5, 9 and 10 are each broken into two parts as a and b. The HPin towards the cytosolic side is connected to helix H4c to one side and TM5 on the other side. Similarly, HPout coming out from the periplasm is connected to the TM10 and H9c. The hairpin along with the broken helix (intramembrane loop) plays a chief role in membrane transport. The NaCT (VcINDY) has two repeated folds with 26% similarity in amino acid sequence from the N-and C-terminal side i.e., TM2-TM6 from the N terminal side is related with TM7-TM11 of C terminal side. The binding pocket for the citrate is formed from the residues HPin, TM5, HPout and TM10. The citrate molecule binds between Na1 (N terminal side hairpin tip-capping loop motif formed by HPin tip, TM5a and TM5b) and Na2 sites (C terminal side hairpin tip-capping loop motif formed by HPout tip, TM10a and TM10b)[12] Figure 2 . The zebrafish Na+ coupled citrate transporter was cloned showing 61% identical and 77% similarity to the human NaCT. The cloned human NaCT showed 77% identity with rat NaCT and the gene is located on human chromosome 17 at p12-13 [13]. Khamaysi et al., studied the slc13 transporter H4c as a dynamic anchor domain which controls the metabolite transport (succinate and citrate). It was observed that intracellular determinants interact with the H4c domain and control the transport. The interaction to both the intracellular determinants and the membrane phospholipids is attained by a sole basic residuei.e ., Arg108. The conclusions were drawn by carrying out several experimental studies in Xenopus oocytes and fluorescent microscopy of mammalian cells. The Arg108 present in highly conserved H4c was found to be critical for the metabolite transport. Neutralization of this basic residues inhibited the transport functions [14].
Figure 2: A NaCT representation of a VcINDY in which transport domain is represented by the binding sites hairpin loops and unwounded helices 5 and 10. The N terminal end is related to the C terminal end through an inverted repeat symmetry (shown in red and blue color box) except the TM1[12,15].
The receptors of the VcINDY can be expressed in two forms - the outward facing state and the inward-facing state for the transport mechanism to occur. The substrate binds to the outward facing state formed by the transport domain (shown in orange) which slides 15Å downward (translocate) with 43o rotation to form the inward facing states (as such elevator is sliding downwards). The elevator is formed by the transport domain and the hydrophobic barrier provided by transmembrane 4 and 9 (oligomerization domain) and scaffold domain. This leads to the release of the substrate to the cytoplasm. After releasing the substrate, the transporter again returns to it outward facing state to start the next cycle [15] (Figure 3) . It was also observed that crosslinking VcINDY to either inward or outward facing state inhibit the transport activity. The test was done by reconstituting the double cysteine mutants in proteoliposomes and access the transport abilities of the transporter. The crosslink was induced with excess HgCl2 in both cases of outward and inward stabilizing mutants. The transport activity was restored when the crosslinking was reduced. The transport activity was strong in HgCl2 treated cysteine less proteins [15].
Figure 3: Substrate transport by elevator type mechanism. The substrate binds to the outward facing of the transport domain, translocation inhibited by the hydrophobic barrier (scaffold and oligomerization domain). The transport domain translocated to 15Å downward rotating 43o to the inward facing state releasing the substrate to the cytoplasm [16].
Although the receptors functional properties are extensively discussed but the different analogues bindings within these receptors for the treatment of various metabolic disorders has not been covered in detail in previously published reviews. This article encompasses different key intermediates analogues responsible for the blocking of these SLC family receptors for promising results.
  1. Distribution and functionThe sodium-coupled with anionic transporters includes three transporters of dicarboxylates and tricarboxylates (NaDC1, NaDC3 and NaCT). The metabolic intermediates of TCA cycle such as citrate, α-ketoglutarate and succinate are the substrate for these transporters which maintains the levels of these intermediates in plasma, urine, and tissue levels [17].
  2. SLC13A5 (NaCT): These receptors are widely distributed. NaCT found in the liver, brain, and salivary glands primarily transport citrate into these tissues. It is major carrier of citrate in the liver [18]. The NaCT (SLC13A5) is mammalian homolog of the Drosophila INDY. The partial loss in the citrate transport causes increase in lifespan in Drosophila [11]. The rat NaCT (Na+ coupled citrate transporter) has 77% similarity with the cloned human NaCT. It mediates the Na+ coupled citrate transport and is the first transporter recognized in the mammalian cells that showed higher affinity for citrates [19]. It is mostly expressed in liver but also found in brain and testis. The transport is electrogenic whereas its activation showed sigmoidal curve (more involvement of the Na+ ions in activation). The citrates are found in high concentration in the human blood and are involved in the metabolic energy and synthesis of fatty acids and cholesterol [20]. NaCT is more selective to citrate than other dicarboxylates or tricarboxylates [11]. The primary neurons culture of mouse cerebral cortex showed the NaCT transport system, but it was found to be absent in the astrocytes. It is Na+-dependent, Li+-sensitive inhibited by the unlabeled tricarboxylate citrate and other dicarboxylates e.g., malate, fumarate, succinate, and α-ketoglutarate [21]. The mutation in SLC13A5 gene codes for Na+/citrate co-transporter results in loss of function and causes neurological disorders that includes neonatal epilepsy, delayed brain development and tooth dysplasia in children. Targeting the NaCT deletion in mice has beneficial effect against diet induced obesity with no brain dysfunction. Mutation different from the brain located in SLC13A5 may have beneficial effect. Thus, there is greater emphasis in designing inhibitors which specifically cause the NaCT loss in the liver rather than NaCT loss in the brain [11]. The SLC13A5 mutation in nine epilepsy patients were identified. The drug acetazolamide (carbonic anhydrase inhibitors) and the atypical anti-seizures drugs decreases the seizures in four patients. Also, it was noticed that the ketogenic diet and fasting worsens the symptoms. The mutations of NaCT transport function and protein expression were further studied by transient transfection against COS-7 cells. It was observed that there was no transport activity from the mutant transporter proteins affecting helix packing or substrate bindings. Other treatment such as chaperones and low temperature were not able to improve the transport functions of mutated NaCT. A decreased protein expression and activity in wild-type transporter have been observed when both the NaCT and mutant NaCT are co-expressed [22]. Selch et al., studied the mutation associated with the SLC13A5 gene NaCT mediate transport functions. Taking HEK293 as wild type and eight mutation NaCT proteins, it was observed that proteins were synthesized identical to the wild types but for the mutants pG219R, pG219E, pT227M, pL420P and pL488P the citrate transport was completely inhibited. These conserved amino acids are present in the transmembrane pore and are important for the NaCT-mediated transport [23]. The VcINDY homolog of SLC13A5 represent the receptors for information on substrate binding sites and mechanism. It has large open cavity towards the cytosolic sides for sodium driven succinate transport and is inhibited by the dicarboxylates such as malate and fumarate. In this it requires four sodium to couple with the substrate (4:1). The NaCT has many functions to perform such as energy production (mitochondria Kreb’s cycle), glycolysis, gluconeogenesis, fatty acid, and cholesterol synthesis in the cytosol. The SLC13A5 also regulates fibroblast growth factor receptors (FGF23) function leading to the overproduction of the FGF23 and abnormal mineralization of the extracellular matrix leading to hypophosphatemic rickets disease. The NaCT activity is stimulated by the presence of Li+ ions [10].
  3. Citrate transporter of Lactic acid bacteriaThe human sodium-dependent citrate transporters (NaCT) and succinate/dicarboxylate from Lactobacillus acidophilus has great similarity in the structure as VcINDY. All these results support for the VcINDY protein structure representing the overall protein structure in ion transporter superfamily[8]. The citrate transporter and the malate transporter of lactic acid bacteria Leuconostoc mesenteroides and Lactococcus lactis were analyzed and both showed transport of substrate containing 2-hydroxycarboxylates. The structural changes either of the OH and COO- drastically reduced the affinity to the transporters. The dicarboxylate with the formula OH-CR-(COO-)2, theS -enantiomer of which binds efficiently and translocated whereas the R -enantiomer has no affinity. The binding order magnitude of citrate or S -enantiomer is one order higher than other uncharged R groups containing compounds including lactate; this shows the importance of second carboxylate in greater affinity with the proteins. The binding preference for theR -enantiomer is more in Leuconostoc mesenteroideswhen R groups are hydrophobic whereas onlyS -enantiomer is translocated in Lactococcus lactis[24]. The 46 residues at the C-terminal region containing C-terminal putative transmembrane XI was analyzed for the substrate recognition through chimeric transporters. The replacement of the C-terminal region in Leuconostoc mesenteroides with Lactococcus lactis and vice versa did not alter the transport characteristics of the di and tri carboxylate i.e ., malate and citrate whereas monocarboxylate substrate mandelate and 2-hydroxyvalerate interactions with the proteins were changed. The incorporation of C-terminal residues of one bacterium into another and vice versa led to higher affinity. The interchanging of the C-termini has more complex effect on R enantiomers. It was indicated that binding pocket is located in other transmembrane and transmembrane XI [25]. The substrate transported by citrate transporter of Leuconostoc mesenteroides and malate transporter of Lactococcus lactis are shown inFigure 4 .Figure 4: The substrate transported with citrate transporter of Leuconostoc mesenteroides and malate transporter of Lactococcus lactis
  4. Citrate Transporter of Klebsiella pneumoniae
The loops of the transmembrane segments VIII and IX (AH loop) represent the amphipathic surface helix of Klebsiella pneumoniae . The mutation of the cysteine residues especially G324C, F331C, and F332C has very low citrate transport activity whereas other mutants I321C and S333C showed decreased activity with membrane permeable thiol reagentN -ethylmaleimide (NEM) but not with membrane impermeable 4-acetamido-4’-maleimidylstilbene-2,2’-disulfonic acid and [2-(trimethylammonium)ethyl]methanethiosulfonate. The impermeability of the reagent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid supports for the localization of the AH loop in the cytoplasmic region [26].
Inhibitors of NaCT
Higuchi et al., showed that deletion of the genes that encode for SLC13A5 in mice protects against obesity and other metabolic disorders such as diabetes. Suppressing the transporter also suppresses the hepatocellular carcinoma. The author reported one irreversible and non-competitive inhibitor of human NaCT, and which does not affect the mouse NaCT. The compound 1 inhibits the constitutively transporter in HepG2 cells and ectopically expressed in HEK-293 cells. The inhibition is in nanomolar range of ⁓100 nM (Figure 5 ). The molecular modelling studies generated human NaCT and mouse NaCT taking humanized variant of VcINDY as the template. The compound and the citrate showed different inhibitors to human NaCT versus mouse NaCT [27].
Figure 5: The compound inhibits HepG2 cell transporter and ectopically expressed HEK-293 cells.
It was further evidenced that HepG2 cells represent the excellent cell models to study the regulatory mechanisms of endogenous di and tricarboxylates. Also, it was found that there is higher rate of succinate transport as compared to citrate. The human retinal pigments epithelial cells transfected with hNaDC-1, hNaDC-3 and hNaCT were compared with citrate and succinate in HepG2 cells and the results showed that HepG2 cells transport were constant with hNaDC-3 and hNaCT expressed in human retinal pigments epithelial cells. The high affinity transport for succinate is consistent with hNaDC-3 [2]. The blockade of hepatic extracellular citrate uptake through the blockade of NaCT may be regarded as potential treatment of metabolic disorders. This led Huard et al., in 2015 to identify and characterize some novel small dicarboxylate molecules especially the active (R )-enantiomer 2 to be highly selective for NaCT compared to NaDC1 and NaDC3. The (S )-enantiomer was found to be inactive. The mechanism for the compound 2 was found to be competitive to the citrate, specific, stereo sensitive and direct to the NaCT receptors. The compound acts a substrate for active cellular uptake and inhibits both in vitro and in vivo by directly binding the compound to NaCT receptors. The IC50 of the compound2 against HEKNaCT was found to be 0.41 µM as compared to the racemate having IC50 value of 0.80 µM[18] Figure 6 .
Figure 6: Compound 2 with their IC50value against HEKNaCT
The inhibition of sodium citrate transporter NaCT (SLC13A5) is a major target for metabolic disease. As discussed above citrate acts as signaling molecules that regulates fatty acid and glucose metabolizing enzymes. Two compounds 3 and 4 inhibits the human NaCT with very high affinity. Compound 3 is very specific for hNaCT whereas, compound 4 inhibits all the three transporters (NaCT, NaDC1 and NaDC3) but NaCT with greater affinity. Compound 3inhibited the citrate uptake (hNaCT) with no inhibition of citrate C14 transport activity in NaDC1 and NaDC3 whereas 4 inhibited all the three transporters at 100 µM. The citrate uptake blockade by compound 3 in the liver inhibits the conversion of extracellular citrate to tricarboxylic acid cycle (TCA) intermediates. This results in reduction of high plasma glucose concentration in mice fed with high fat (Figure 7 ). Pajor et al ., 2016 explored the molecular modelling inside the citrate receptors and analyzed the amino acids involved in the binding sites. The amino acid residue (G228, V231, V232, and G409) near the citrate binding sites affect both the transport as well as inhibition of citrate uptake. The amino acid V231 distinguish between the compound 3 and4 as inhibitors. Some of the residue outside the binding sites such as Q77 and T86 are important in NaCT inhibition[28].
Figure 7: Sodium citrate transporter NaCT (SLC13A5) inhibitors
One of the studies carried out by Huard et al., optimized the earlier series of synthesized compound to more potent compound5 . The compound showed potent inhibition of the [14C] citrate uptake in the liver and kidney. The compound showed more suitable in vivo pharmacokinetic profile for better in vivo pharmacodynamics. The compound also reduced the plasma glucose concentrations[18] (Figure 8 ).
Figure 8: Optimization of the compound 1 to more active isomer 5 against NaCT receptors
It was demonstrated that metformin inhibits the NaCT plasma membrane citrate transporter in HepG2 cells with decrease in the levels of the citrate and decreases mRNA levels. It inhibits gluconeogenesis and thus has anti-diabetic effects. An 5’ adenosine monophosphate-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) has similar effect. Activating AMPK inhibits mTOR which further inhibits rapamycin and thus decreases NaCT expression. The decrease in the cellular levels of the citrate stimulate glycolysis and inhibits gluconeogenesis (citrate suppresses glycolysis by inhibiting phosphofructokinase-1 and activate gluconeogenesis by activating fructose 1,6-biphosphate) [29] (Figure 9 ).
Figure 9: Flow diagram showing new mechanism of action of the metformin acting on SLC13A5 citrate receptor
The cytosolic citrate activates fatty acid synthesis and downregulates the glycolysis and β-oxidation of fatty acids. The cytosolic concentration of the citrate controls rate of synthesis of fatty acids in the liver and adipose tissue which in turn is controlled by the transport through NaCT transporter. It was evidenced that mutation in homologous fly gene results in reduced fat storage. The study in the mice (NaCT knockout) further confirms reduced lipogenesis, higher lipid oxidation and energy expenditure. This led to protection of mice from obesity and insulin resistance [12]. The divalent anion sodium symporter (DASS) includes Staphylococcus aureusNa+/dicarboxylate symporter SdcS, mammalian SLC13 Na+/dicarboxylate cotransporters NaDC1 and NaCT. The anthranilic acid derivatives such as N -(p-amylcinnamoyl) anthranilic acid 6 is a slow inhibitor of the mammalian members. The SdcS was also inhibited by the N -(p-amylcinnamoyl) anthranilic acid and nonsteroidal anti-inflammatory drugs (NSAIDs) such as flufenamate and niflumate as allosteric inhibitors. The IC50 was calculated as 55 µM forN -(p -amylcinnamoyl) anthranilic acid (Figure 10 ). With succinate the graph was sigmoidal with K0.5 of 9 µM and Hill coefficient 1.5. When given withN -(p -amylcinnamoyl) anthranilic acid K0.5remains to be 1.5 but there was decrease in the V max and increased in Hill coefficient. Also, the mutants (N108C) reactivity was not affected by the N -(p -amylcinnamoyl) anthranilic acid inhibition [30].
Figure 10 : The N -(p-amylcinnamoyl) anthranilic acid inhibitors of both mammalian and Staphylococcus aureusNa+/dicarboxylate symporter
The classical inhibitor benzene tricarboxylate for the mitochondrial citrate transporter protein led Aluvila et al., to identify competitive inhibitors against the mitochondrial citrate transporter protein through in silico screening of the ZINC database. A total of 10 compounds were identified which showed more than 50% inhibitions at the concentration of the 1 mM. One of the compounds showed the highest percentage inhibition of 85% as compared to the benzene tricarboxylate (% inhibition = 79%). Figure 11 shows the compound 7 identified by the in-silico database screening taking benzene tricarboxylate as the reference. One of the compound 8 acts as potent inhibitors against both the bacterial citrate transporter protein as well as plasma membrane citrate transporter [31].
Figure 11: Competitive mitochondrial citrate inhibitors byin silico screening of the ZINC database.
Na+/Dicarboxylate co-transporter, SLC13A2 (NaDC1) : The hNaDC1 found in renal proximal tubule and small intestine and has low affinity for both the di and tri-carboxylate (oxaloacetate, fumarate, and malate). The main substrate is succinate (Km value of 0.9 mM), α-ketoglutarate and citrate (main physiological substrate). In renal proximal tubule and small intestine, it reabsorbs the intermediates of the TCA from urine and diet, maintains the urinary levels of citrate and plays a crucial role in the development of kidney stone [10,17]. Citrate can chelate the calcium (Ca2+) and avoid its precipitation. Thus, low urinary citrate promotes the development of the kidney stones [3]. It involves bindings of three sodium ions and then the dicarboxylate ions results in passage of one positive ions. The ion lithium competitively inhibits the transport after binding at one of the sodium bindings sites, but the sensitivity varies with species-to-species [10]. Human NaCT is activated by the presence of Li+ whereas rodents NaCT showed inhibition[13]. The stimulation of NaDC1 by metabolic acidosis through physical exercise, clinical treatments led to decrease in the citrate concentration [10]. The transporter of SLC-13 family transport di or tricarboxylate (succinate or citrate) along with two to four Na+ ions. Colas et al., in the year 2017 modeled outward facing conformation of Na+/dicarboxylate co-transporters of both human (h) and rabbit (rb) using outward facing model of bacterial homolog VcINDY template. It was observed that mutagenesis in rbNaDC1 of T474 with cysteine results in inactive protein. The cysteine substitution in M539C results in low affinity to both sodium and lithium ions, indicates for the hNaDC3 sites. The mutants of Y432C and T86C led to increased Km value for succinate suggesting for these amino acids in outward-facing substrate binding sites[32].
Colas et al ., 2015 uses homology model for human hNaDC1, mouse mNaDC1 and mouse mNaDC3 and human hNaCT and predicted small molecules inhibitors by the virtual screening. The compound 9 was selected for generating the most potent hit. The compound 9inhibited [14C] transport activity in all the transporters except for NaCT. All the five compounds (10-14 ) inhibited one or other SLC13 members, compound 11 being the most potent inhibitors in mNaDC1 (IC50 = 72 µM in one experiment and 82 µM in second experiment) and the compound 15most selective against hNaDC3 inhibition (cis -inhibition assay). The IC50 value of 11 against mNaDC1 was 85 µM (one experiment) and 100 µM in second experiment whereas it poorly inhibited the hNaDC3 (⁓2 mM). The compound 11 was found to show the bindings with amino acid sidechains Thr236 of mNaDC1 and was specific to this transporter. The hNaCT binds with citrate and four sodium ions inhibited by the dicarboxylate 13 . The newly compounds showed inhibition at IC50 value in µM range[1] (Figure 11 ).
Figure 11: Compounds representing the SLC13 members inhibitors;15 being the most selective against hNaDC3
SLC25A1/Citrate/isocitrate Inhibitors
Cytosol citrates provide the source of acetyl CoA for fatty acid and sterol biosynthesis and act as regulators controlling gluconeogenesis, glycolysis, and lipogenesis. Conversely citrate/isocitrate carrier promotes citrate to the mitochondria from the cytoplasm thus causing TCA and oxidative phosphorylation with the generation of NADPH. This reverse effect led to the cancer cell expansion and growth of the tumor cells. Undoubtedly citrate/isocitrate carrier utilizes the resources to meet the energetic demand whereas the loss of it is pathogenic. Benzene tricarboxylate 16 (1st generation; analogs of citrate) interferes with the citrate bindings proteins at high concentration (5 mM) acts as citrate transporter protein inhibitors (CTPI). The second inhibitors CTPI-1; 17 was effective also at higher concentration of 1-2 mM and acts as competitive inhibitor. There is 20-fold improvement in the activity with CTPI-2; 18 as compared to CTPI-1; 17 and inhibits the CIC at a lower dose of 10-50 µM. Also, CTPI-2; 18 reverts steatosis, reduces obesity, glucose intolerance, triglycerides, and cholesterol levels [35].Figure 12 showed the structure of first, second and third generation of citrate/isocitrate inhibitors. High levels of SLC25A1 showed enhanced levels of immune checkpoints inhibitors for antibody targeting and pro-inflammatory response for tumor killings. The inhibition of SLC25A1 reduced gluconeogenesis (involved in regulation of blood glucose levels) and thereby normalizes the hyperglycemia and glucose tolerance [36]. The SLC25A1 specific CTPI-2 inhibitors reduces macrophage infiltration, preventing steatohepatitis, revert symptoms of inflammatory steatohepatitis and also modify obesity related with high fat. Inhibition of SLC25A1 inhibits peroxisome proliferator-activated receptor gamma (PPARγ) which inhibits the gluconeogenic genes and thus normalizes the hyperglycemia and glucose intolerance [37].
Figure 12: First, second, and third generation citrate and isocitrate inhibitors
Citrate role in Cancer
The citrate exists as an essential molecule in the metabolism and its excess availability by the citrate transporters causes high growth and proliferation of cancer cells. Inhibiting the mitochondrial and the cytoplasm citrate transporters may provide a viable strategy to develop chemotherapeutic agents [38]. The plasma membrane citrate transporter may act as tumor marker and can help in diagnosis of cancer. Glutamine is a major source of citrate in the cancer cells as the citrate produced either by the Krebs cycle in mitochondria or by reductive carboxylation of glutamine. The mitochondria activity is reduced in cancer, so the major source of citrate synthesis is through increase reverse activity of the Kreb’s cycle. The plasma membrane citrate transporter may be considered as a novel target for the cancer identification and treatment [39]. The extracellular citrate promotes the cancer metabolism by reversing the Kreb’s cycle from glutamine. It is very much possible to be observed in citrate rich organs such as liver, brain and bones where it may enhance the secondary tumor growth, metastasis and in colonizing the cancer cells survival [40]. The cancer cells have increased levels of the ATP citrate lyase than normal cells such as colorectal, breast, non-small lung cell and hepatocellular cancers, etc. Overexpression of the ATP citrate lyase may be inhibited by either chemical inhibitors or knockdown of ATP citrate lyase [41]. Studies have demonstrated that increase in the citrate or inhibition of ATP citrate lyase stop the tumor growth, inhibit the key anti-apoptotic Mcl-1, and increase in cisplatin sensitivity. Reduced formation of acetyl CoA from decreased citrate concentration causes deacetylation of the proteins and inhibition of apoptosis and epigenetic changes thereby resulting in cancer aggressiveness [42]. The decreased acetyl CoA (reduced intracellular lipid levels and histone acetylation) was also observed viacaspase-10-mediated cleavage of ATP citrate lyase. The decreased histone acetylation downregulates metastatic and proliferative genes [43].In vitro studies showed that cancer cells deprived of citrate with specific inhibitor gluconate (plasma membrane citrate transporter) inhibit the metastases and reduces the growth of human pancreatic cancer cells and angiogenesis in vivo . The above findings provide evidence that citrate metabolite is an important support for the progression of cancer [44]. Results of some preclinical studies showed that high doses of citrate exhibit anticancer effects that includes inhibition of signaling pathway (IGF-1R/AKT), inhibition of growth of tumors, and promotion of apoptosis [45]. The high dose of citrate inhibited the A549 lung cancer growth. The increased citrate concentration makes tumor cell differentiation, higher infiltrating T-cells, increased cytokines and inhibited IGF-1R-AKT-PTEN-elF2a phosphorylation. Both the glycolysis and the TCA cycle were found to be suppressed in vitro [46]. The cancer cells have more citrate oxidizing potential as compared to the normal prostate cells. The level of the citrate gets decrease while there is an increase in the levels of the choline moving from normal cells to the cancerous prostate analyzed by magnetic resonance spectral imaging [3]. Cancer cells development causes extracellular acidity which in turn makes many anticancer drugs ineffective as acidity promotes protonation and decreases internalization. The phosphofructokinase-1 (PFK1) and PFK2 (regulator of the glycolysis) may be inhibited by high concentration of citrate as these kinases enhances the glycolysis, signaling cascade and cell cycle progression against in vitro studies in HCC cell lines. These inhibition decreases the ATP in the cells, acts against the HIF-1α and PI3K/AKT signaling. This promotes apoptosis, increase in the action of cisplatin treatment, and inhibits the cancer cell growth [47].
  1. Citrate role in InflammationCitrate/isocitrate may increase inflammation and thus inhibiting the citrate/isocitrate reduces the citrate transport and hence decrease levels of pro-inflammatory prostaglandins E2 (PGE2) and nitric oxide (NO; mediators of the inflammation response) [35]. Citrate is considered to produce pro-inflammatory molecules. Excess citrate gets cleaved into acetyl-CoA and oxaloacetate by ATP citrate lyase in the cytosol after translocating from the matrix via mitochondrial citrate carrier. Acetyl CoA produces NADPH for NO and ROS production by using PGE2 and oxaloacetate. Itaconate derived through citrate results in the synthesis of inflammatory mediators and thus inhibiting either citrate or the ATP citrate lyase can results in reduction of inflammatory mediators [48]. Infantino et al ., in a study showed that citrate carrier has a role in the inflammation as citrate carrier mRNA and protein increase the lipopolysaccharide (LPS)-activated immune cells thus citrate carrier gene silencing and its inhibition significantly reduce nitric oxide and ROS and prostaglandins [49]. The inhibitors of ATP citrate lyase; tricarballylic acid could reduce the citrate induced TNF-α as ATP citrate lyase increased the citrate metabolism. The tricarballylic acid analogue of citrate has lower affinity to chelate with calcium and thus able to block ATP citrate lyase more effectively thereby reducing the augmentation efficiency of the citrate to LPS induced TNF-α inflammatory response [50]. In LPS stimulated RAW 264.7 macrophages calcium citrate treated cells showed reduced intracellular ROS and increased in activities of antioxidant enzymes. It also inhibits the NO production and other pro-inflammatory markers such as iNOS, COX-2 and NF-kB. The pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) were also reduced indicating that calcium citrate has anti-inflammatory properties [51]. There was higher expression of SLC25A1 (mitochondrial citrate carrier) and ATP citrate lyase in Bechet’s syndrome patients (multisystemic inflammatory disorder). Mitochondrial citrate or its metabolites such as acetyl-CoA and OAA are important mediators of inflammation [52]. Citrate metabolism into acetyl-coenzyme A is utilized for fatty acid synthesis and protein acetylation which in turn are linked with macrophage and active dendritic cells activation. One of the inhibitors itaconate or methylene succinate possesses antibacterial and anti-inflammatory agents by inhibiting succinate dehydrogenase [53]. Figure 13 showed the structure of itaconic acid or methylene succinic acid.Figure 13 : Structure of itaconic acid or methylene succinic acid
  2. Citrate role in CNS physiologyThe astrocytes in the CNS produce a large amount of citrate. It can be reflected by the high concentration of the citrates in cerebrospinal fluids (CSF). The human SLC13A5 loss causes severe epilepsy and encephalopathy. This is not obligatory as SLC13A5-knockout mice do not show any evidence of epilepsy or encephalopathy [54]. In another study however it was confirmed by Henke et al ., that SLC13A5 deleted mouse showed increased frequency of seizure (observed in the 50% of the mice and low frequency in other 50%), alteration in the citrate concentration in CSF (lower than in the plasma but mutant mice citrate is higher than wild types) and brain tissue and pro-epileptogenic excitability changes in the hippocampus. The generalized seizure is mainly associated with the knockout mice. The histological changes in hippocampus and para hippocampus do not show any neuronal changes in either wild or knockout mice [55]. The overexpression of the SLC13A5 neuron-specific receptors in mice showed autistic-like behaviors. It also showed the disrupted white matter integrity and different synaptic functions and structures. The hippocampus and the cortex area showed exceptional adaptation in the complete sets of proteome and acetyl proteome. The study showed that there is clear link between the aberrant citrate/acetyl CoA flux and autistic-like behaviors [56]. Citrate has a role to chelate the divalent cations including Ca, Mg, and Zn and thus regulate the extracellular concentration of these ions for excitation in the neurons and further acts as modulator of glutamate receptors and NMDA subtypes of receptors in CNS. An increase in citrate or decrease in the glutamine concentration was confirmed both in vivo andin vitro with use of fluorocitrate which led to the inhibition of the TCA cycle and loss of GABA synthesis. There is reported correlation of increased CSF glutamine and Mg2+concentration in depressed patient [57]. It also showed hyperfunction of NMDA receptor signaling through some literature studies. This can be better understood by the zinc chelation hypothesis. The SLC13A5 mutation causes extracellular citrate to rise which then start chelating zinc. Negative allosteric regulation of the NMDA receptors is lost with large Ca2+ influx and that leads to enhance NMDA receptor mediated synaptic transmission and pro-convulsant effect [54] Figure 14.Figure 14: Zinc chelation hypothesis; the normal SLC13A5 receptor transport of extracellular citrate to cytoplasm takes place, the NR2A subunits of NMDA receptors occupied by the zinc ion regulate the Ca2+ influx. Mutated SLC13A5 led to increase in the extracellular citrate, chelates the zinc and allosteric inhibitors to NMDA receptors is freed led to increased Ca2+influx and enhanced NMDA receptor-mediated synaptic transmission. The SLC13A5 deficiency dysregulate the hepatic metabolism and initiate the pediatric epilepsy. Kumar et al., used the13C isotopes to study the pathophysiology of the disease against SLC13A5 deficient hepatocellular carcinoma cells and primary rat cortical neurons and found that hypoxic HCC cells use extracellular citrate for fatty acids and TCA intermediates. Under limited conditions of glutamine/other nutrients and oxygen conditions, the citrate supports the growth and lipid synthesis of HCC cell lines. Also, it was evident that citrate uptake in Huh7 cells is protective against Zn2+ cytotoxicity [58]. The congenital disorder 22q11.2 deletion syndrome was studied by Napoli et al . and found to have serious role in the mitochondrial citrate transporter SLC25A1. There were metabolic differences with dysregulation in energy homeostasis in 22q11.2 deletion syndrome children. The children showed elevated levels of cholesterol and 2-hydroxyglutaric acid (2HG) and less than half were also associated with hyperprolinemia. It was also observed that there was a shift from oxidative phosphorylation to the glycolysis in 22q11.2 deletion syndrome children and increase in reductive metabolism of α-ketoglutarate and glucose as carbon source for TCA cycle [59].
  3. Role of citrate in fatty liver disease
The SLC13A5 receptors present in the sinusoidal membrane of the hepatocyte’s uptakes the circulating citrate for the metabolic use [54]. It was evident that dysregulated expression of ATP citrate lyase was found in the patient with non-alcoholic fatty liver disease (NAFLD; associated with lipid accumulation). The lipid accumulation increases the endoplasmic reticulum (ER) stress and upregulated ATP citrate lyase expression through internal ribosome entry site mediated translation. The translation of mRNA is Cap independent and stimulatede novo lipogenesis [60]. Guo et al., showed that acetylation of the ATP citrate lyase at Lys-540, Lys-546 and Lys-554 (3K) increases protein stability and promotes cell proliferation and lipid synthesis in the lung cancer cells. The increased acetylation of 3K and decreased SIRT2 levels were also found in both livers of the mice and the human with NAFLD. Overexpression of hepatic SIRT2 decreased the acetylation of 3K and protein levels and thereby lessen the hepatic steatosis in high fat/high sugar diet-fed mice[61]. Dysregulation of hepatic ATP citrate lyase led to hepatic steatosis, insulin resistance and hyperglycemia. It was evaluated in leptin-deficient db/dbmice and was found that ATP citrate lyase is selectively elevated in the liver and not in white adipose tissues. Abolition of ATP citrate lyase reduced the hepatic content of acetyl CoA and malonyl CoA and inhibited the de novo lipogenesis in hepatic steatosis in db/dbmice. It also inhibited nuclear hormone proliferator-activated receptor-gamma coactivator (PPAR) γ receptors. In hepatic abrogated ATP citrate lyase improves systemic glucose metabolism by down regulated expression of gluconeogenic genes in the liver and enhanced insulin sensitivity [62]. Increased citrate levels were observed in the NAFLD may be due to increased fatty acids. The excess citrate excites hydrogen peroxide (hydroxy radicals) induced oxidative stress in HepG2 cells [63]. The cytoplasmic citrate by plasma citrate transporter (SLC13A5), ATP dependent lyase (ACLY), and mitochondrial citrate regulate de novo lipogenesis. The chronic liver disease caused by NAFLD progress due to upregulation of the de novo lipogenesis. Curcumin was investigated against NAFLD induced by oleic acid and palmitic acid in primary mouse hepatocyte and with high-fat and high-fructose diet. Curcumin was found to reduce oleic acid/palmitic acid or high fat and high fructose induced NAFLD (hyperlipidemia and hepatic lipid deposition). Curcumin may block the transporter SLC13A5, SLC25A1 and enzyme activity of ACLY and improves the metabolic lipid disorder. The mechanism by which curcumin acts is by blocking the citrate transport (improves dysregulated SLC13A5) and inhibitory effect of ACLY (cleaves citrate into acetyl-CoA and oxaloacetic acid) in oleic acid and palmitic acid stimulated HepG2 cells via AMPK-mTOR signaling pathway. Curcumin has significant lipid lowering effect and inhibit the overexpression of SLC13A5 and ACLY. This further reduces the upregulation of de novo lipogenesis and regulates the lipid accumulation in NAFLD in mice [64]. The hydroxy citric acid extract from the Garcinia cambogia was evaluated for the inflammatory, atherogenic and metabolic biomarkers in 40 obese women with NAFLD. The results showed decrease in the macronutrient intake and energy in both the hydroxy citric acid group and the control groups. There was also reduction in the levels of the triglycerides, total cholesterol, fasting blood sugar, low density lipoproteins cholesterol (LDL-C) and increase in high density lipoprotein cholesterol (HDL-C) in the hydroxy citric acid. The ratio of TG/HDL-C ratio was decreased as compared to the control. The inflammation factors were not reduced as such in the hydroxy citric group but it improved metabolic factors [65].
Role of citrate in the treatment of obesity and type 2 diabetes mellitus (T2DM)The ATP citrate lyase, the one converting citrate to acetyl CoA, is highly induced in the obese patient with chronic kidney disease. The increase activity of ATP citrate lyase leads to the ectopic accumulation of lipid, glomerulosclerosis, and albuminuria. It also upregulates many rate-limiting lipogenic enzymes and promotes de novo lipogenesis for lipid accumulation. The raised acetyl CoA also causes hyperacetylation at the sites of H3K9/14 and H3K27. The citrate inhibitor completely blocked the hyperacetylation as well as induction of lipogenic enzymes. The ATP citrate lyase promotes renal lipid accumulation, fibrogenesis and renal injury in obesity [66]. A clinical study was conducted for 3 months in 100 obese patients to evaluate the effects of hydroxy citric acid and one of the major ingredients of Garcinia cambogia extract. Study results showed that there was significant reduction in the body weight, serum triglycerides, HDL, and LDL levels. The hydroxy citric acid treatment may reduce the body weight gain and fat deposits in obese patients [67]. The enzymatic activities and proteins levels of the citrate synthase were reduced in obese patients in another study of human omental adipose tissue [68]. The increased activity of the ATP citrate lyase in the livers of the hereditary obese mice is two to four times more than non-obese siblings and the activity was observed to be reduced in starvation and increased by refeeding. The increased in activity is also found to be increased after the onset of lactation [69]. The hypothesis that exogenously taken citrate in the form of processed foods and drinks contribute to the weight gain was evaluated in mice fed with citrate with or without sucrose. The results showed that neither it increased the weight gain nor change the lipid pattern in any of the tested groups (citrate or citrate + sucrose). The group with citrate + sucrose enhanced the pro-inflammatory cytokines (TNF-α, IL-6, IL-10 and IL-1β), fasting glycemia and glucose intolerance. Thus, citrate consumption may act as contributing agent for the complication associated with obesity such as altered glucose metabolism or adipose tissue inflammation [70]. Patients with obesity and diabetes mellitus are more likely to be associated with low urine citrate excretion among nephrolithiasis (15% more than 7% control). Uric stone and calcium oxalate monohydrate are two most common stone in citrate wasting groups (low urine citrate excretion). It was evident that citrate wasting groups showed higher uric acid, calcium, oxalate, and sodium [71]. In diabetes patients, the enzymatic activity was reduced rather than proteins levels. The key enzymes of the TCA cycle (citrate synthase) were affected. The compounds shown in the Figure 15 with the general formula19 claims for treating diseases related with the citrate transporter. The compound is used for treating diabetes especially T2DM, obesity, and other age-related disorders. Among them the compound 20 and 21 showed highest functional activity against citrate INDY in HEK293 and HepG2 cell lines [72]. The low citrate synthase activity as tested in A/J strain variant of H55N polymorphism accumulated more fats and was tolerant to glucose. The knockdown citrate synthase activity accelerated palmitate induced apoptosis in 50% of the tested in C2C12 mouse muscle cells [73]. Citrate administration could also decrease diabetic adverse cardiac changes. It decreased mean glucose levels whereas glucose levels are less affected in streptozotocin induced diabetic rats. Citrate application with the antioxidants decreased blood glucose levels protecting the β-cells of the pancreas from damage. The citrate groups reduced the harmful effect on the heart tissue of the diabetes. Also, caspase 3 immunoreaction optical density was decreased in citrate group and integrity of the intercalated discs was found to be maintained as compared to the diabetic groups [74]. The rate of citrate transport was reduced to 35% in diabetic mitochondria. There was also an increase in cholesterol contents and ratio of cholesterol/phospholipid ratio. The analysis confirmed the accumulation of hepatic citrate carrier mRNA, and decreased levels of the protein. The lower contents of both the mitochondrial citrate carrier and protein is because of decrease citrate carrier in diabetes [75].Figure 15: General formula and most potent compound for treating type 2 diabetes, obesity, and age-related disorders