From Glucose to Lactate and Transiting Intermediates Through Mitochondria, Bypassing Pyruvate Kinase: Considerations for Cells Exhibiting Dimeric PKM2 or Otherwise Inhibited Kinase Activity

Abstract

A metabolic hallmark of many cancers is the increase in glucose consumption coupled to excessive lactate production. Mindful that L-lactate originates only from pyruvate, the question arises as to how can this be sustained in those tissues where pyruvate kinase activity is reduced due to dimerization of PKM2 isoform or inhibited by oxidative/nitrosative stress, posttranslational modifications or mutations, all widely reported findings in the very same cells. Hereby 17 pathways connecting glucose to lactate bypassing pyruvate kinase are reviewed, some of which transit through the mitochondrial matrix. An additional 69 converging pathways leading to pyruvate and lactate, but not commencing from glucose, are also examined. The minor production of pyruvate and lactate by glutaminolysis is scrutinized separately. The present review aims to highlight the ways through which L-lactate can still be produced from pyruvate using carbon atoms originating from glucose or other substrates in cells with kinetically impaired pyruvate kinase and underscore the importance of mitochondria in cancer metabolism irrespective of oxidative phosphorylation.

Glucose and Lactate in Cancer: Background

It is a well-known fact that most cancers exhibit increased rates in glucose consumption (Bose and Le, 2018). This is clinically exploited by following radionuclide-labeled glucose analogs for the purpose of tumor imaging in living human beings (Feng et al., 2019). The very same cancers are also known to be major lactate producers, which is important for their survival (de la Cruz-Lopez et al., 2019). The combination of an increased consumption of glucose with an increase in lactate output led to the assumption that cancers exhibit an increase in glycolysis; although this is true, serving the purpose of generating glycolytic metabolites which are diverted toward biosynthetic processes (DeBerardinis et al., 2008) and NADPH by the pentose phosphate pathway (Icard and Lincet, 2012), most tumors express a dimeric form of the M2 isoform of pyruvate kinase which has been reported to be much less active than that found in healthy cells; furthermore, numerous posttranslational modifications and mutations have been reported for this gene product, leading to a much reduced activity but still fueling cancer aggression (see section “Pyruvate Kinase”). Even more so, tumor cells with undetectable levels of pyruvate kinase still producing lactate can be found in vivo (Israelsen et al., 2013). On one hand, the decrease in pyruvate kinase activity is important for maintaining a metabolite “traffic jam,” forcing upstream metabolites toward biosynthetic pathways; on the other hand, it points to a metabolic conundrum because L-lactate may only originate from pyruvate, a metabolite arising from phosphoenolpyruvate (PEP) through pyruvate kinase in glycolysis (see Figure 1). The purpose of this review is to highlight the pathways that can lead to pyruvate and lactate—even commencing from glucose—bypassing pyruvate kinase. This is important because (i) carbon-labeled atoms in glucose may appear in lactate without net ATP production from glycolysis and (ii) hints on the possibility that other pathways leading to pyruvate/lactate could be crucial for cancer cell survival that are perhaps amenable to pharmacological and/or genetic manipulation. The list of pathways appearing below has been assembled by mining the following databases: Kyoto Encyclopedia of Genes and Genomes¹ (Kanehisa and Goto, 2000), BRaunschweig ENzyme Database² (Jeske et al., 2019), Metabolic Atlas³ (Robinson et al., 2020), Biochemical, Genetic, and Genomic knowledge base⁴ (King et al., 2016), MetaNetX⁵ (Moretti et al., 2016), Human Metabolome Database⁶ (Wishart et al., 2018), and Virtual Metabolic Human⁷ (Noronha et al., 2019).

FIGURE 1.

Biochemical pathways connecting glucose or other metabolites to pyruvate and L- or D-lactate. The box in magenta represents a mitochondrion. Glycolysis is highlighted in green. Metabolites found both inside and outside the mitochondria that are not connected with an arrow are highlighted in matching striped colors (to avoid arrow clutter). For abbreviations, see Table 1.

Pyruvate Kinase

Pyruvate kinase generates ATP at the “substrate level” in the absence of oxygen by catalyzing the dephosphorylation of PEP to pyruvate (see Figure 1). There are four isoforms denoted as L, R, M1, and M2. For details regarding kinetic properties, tissue distribution, and regulation, the reader is referred to the review by Israelsen and Vander Heiden (2015). In the present review, the PKM2 isoform will be specifically examined; for a more thorough evaluation, the reader is referred to Li et al. (2014, 2018), Wong et al. (2015); Yang and Lu (2015), Dayton et al. (2016b), Hsu and Hung (2018), and Alquraishi et al. (2019). The non-enzymatic functions of PKM2 are examined elsewhere (Hoshino et al., 2007; Stetak et al., 2007; Luo et al., 2011; Yang et al., 2012; Yang and Lu, 2013).

Basically, PKM2 exhibits lower enzymatic activity compared to that by PKM1 (Yamada and Noguchi, 1999) and is allosterically regulated by fructose-1,6-bisphosphate (FBP); it exists either as a dimer with low affinity for PEP or as an FBP-bound tetramer with high affinity for PEP (Mazurek et al., 2005; Zhang et al., 2019). Although PKM2 has been branded as “the predominant isoform in cancer cells” (Altenberg and Greulich, 2004; Mazurek et al., 2005), further scrutiny in 25 human malignant cancers, six benign oncocytomas, tissue-matched controls, and several cell lines showed that “PKM2 dominance was not a result of a change in isoform expression, since PKM2 was also the predominant PKM isoform in matched control tissues.” Therefore, a switch from PKM1 to PKM2 isoform expression during malignant transformation may not be taking place, as previously postulated (Christofk et al., 2008). Mindful of the controversy surrounding the proposed functions of PKM2 (Hosios et al., 2015; Harris and Fenton, 2019), the group of Vander Heiden characterized the effects of cancer−associated PKM2 mutations on enzyme kinetics and allosteric regulation and reported that a decrease in PKM2 activity supports the rapid proliferation of cells (Liu V. M. et al., 2020). This is in line with earlier reports showing that a decrease in PKM2 activity due to posttranslational modifications (Lv et al., 2011) or inhibition by oxidative stress (Anastasiou et al., 2011) promotes tumor growth (Prakasam et al., 2018). Alternatively, exposure to small molecule PKM2 activators or expression of the constitutively active PKM1 thwarts cancer cell proliferation (Anastasiou et al., 2012). Finally, it has been also shown that PKM2 is not even required for the growth of many cancers (Cortes-Cros et al., 2013; Israelsen et al., 2013; Wang et al., 2014; Lunt et al., 2015; Dayton et al., 2016a, 2018; Lau et al., 2017; Tech et al., 2017; Hillis et al., 2018). In aggregate, the consensus seems to be that the lower the pyruvate kinase activity, the greater the stimulation of tumor growth. As discussed in the section below entitled “Evidence Showing That Pyruvate Kinase Inhibition Does Not Lead to a Proportional Decrease in Pyruvate/Lactate Formation,” even those cells exhibiting low—or even undetectable—pyruvate kinase activity still produce lactate, which begs the question: where does this lactate come from?

Evidence Showing That Pyruvate Kinase Inhibition Does Not Lead to a Proportional Decrease in Pyruvate/Lactate Formation

In Cortes-Cros et al. (2013), it was shown that knockdown of both PKM1 and PKM2 (PKM2 knockdown was on the order of > 95%) leading to an approximately fivefold decrease in overall pyruvate kinase activity yielded only a ∼50% decrease in the appearance of ¹³C originating from glucose to lactate.

In Chaneton et al. (2012), silencing of both PKM1 and PKM2 to an extent greater than 90% led to only a ∼30% decrease in pyruvate and lactate production, while PEP concentration increased by 100%.

In Vander Heiden et al. (2010), it was shown that cancer cell lysates expressing no pyruvate kinase activity produced 50% of pyruvate from PEP compared with the total cell lysates. Although in this work it was postulated that phosphate from PEP is transferred to the catalytic histidine on human PGAM1, this claim was subsequently rejected by the same authors, attributing their earlier findings to contaminating ATP−dependent protein kinases (Hosios et al., 2015).

In all of the abovementioned studies, it was assumed that, in view of severely diminished pyruvate kinase activity, pyruvate and lactate production is attributed to carbon sources other than glucose. Indeed Yu et al. (2019), determined that, in pancreatic ductal adenocarcinoma cells with PKM1 and PKM2 knockdown, cysteine catabolism generated ∼20% of intracellular pyruvate. The purpose of the present review is to not only outline these pathways but also show additional ways for obtaining ¹³C labeling in pyruvate or lactate originating from glucose; furthermore, since some of these pathways involve intermediates that transit through the matrix, the role of the mitochondria is emphasized, which is unrelated to the concept of oxidative phosphorylation.

Pathways Leading to Pyruvate Commencing From Glucose: Intermediates Not Transiting Through the Mitochondria

The pathways shown in this section refer to Figure 2 (lavender arrows). Multiple arrows imply multiple biochemical steps.

FIGURE 2.

Pathways leading to pyruvate commencing from glucose, highlighted in lavender: intermediates not transiting through the mitochondria. For abbreviations, see Table 1.

(1) Glc + PEP → Glc-6-P + pyruvate: This reaction is catalyzed by glucose-6-phosphatase (G6PC) (Nordlie, 1974; Colilla et al., 1975) (for abbreviations, see Table 1). In humans, G6PC expression was reported to be elevated in GBM when compared with normal brain (Abbadi et al., 2014), while in rodent hepatomas it was found to be decreased (Weber and Cantero, 1955).

TABLE 1.

Abbreviations.

2-Oxoglrm	2-oxoglutaramate (a-ketoglutaramate)
2-PG	2-Phosphoglycerate
3-OH-pyr	3-hydroxypyruvate
3-PG	3-Phosphoglycerate
4-OH-proline	4-hydroxyproline
5-10 mTHF	5-10 methylene-Tetrahydrofolate
ACLY	ATP Citrate Lyase
ACO	Aconitase
ADH	Alcohol Dehydrogenase
AGXT	Alanine-glyoxylate Aminotransferase
aKG	a-ketoglutarate
Ala	Alanine
ALXT	Alanine-Ketomalonate Transaminase
Aml	Aminomalonate
Asn	Asparagine
Asp	Aspartate
cADC	cis-Aconitate Decarboxylase
CLYBL	Citramalyl-CoA Lyase
CS	Citrate Synthase
CYB5D1	Cytochrome B5 Domain-Containing Protein 1
Cys	Cysteine
D2HGDH	D-2-Hydroxyglutarate Dehydrogenase
DAAO	D-amino acid Oxidase
D-LDH	D-Lactate Dehydrogenase
FAHD	Acylpyruvase
FH	Fumarate Hydratase
Fum	Fumarate
G6PC	Glucose 6 phosphatase
GALK	N-acetylgalactosamine Kinase
GalNAc	N-Acetylgalactosamine
GalNAc-1-P	N-Acetylgalactosamine-1-Phosphate
GAPDH	Glyceraldehyde 3 Phosphate Dehydrogenase
Glc	Glucose
Glc-6-P	Glucose-6-phosphate
Gln	Glutamine
Glu	Glutamate
GLUD	Glutamate Dehydrogenase
Gly	Glycine
Gly-3-P	Glyceraldehyde-3-Phosphate
GLYCTK	Glycerate Kinase
GOT	Aspartate Aminotransferase
GPAT	Glutamine-Pyruvate Transaminase
GPT	Alanine Aminotransferase
GRHPR	Glyoxylate Reductase
HAO	Hydroxyacid Oxidase
IDH	Isocitrate Dehydrogenase
Ile	Isoleucine
KGDHC	a-Ketoglutarate Dehydrogenase Complex
LAAO	L-amino-acid Oxidase
LDH	Lactate Dehydrogenase
Leu	Leucine
Mal	Malate
MDH	Malate Dehydrogenase
ME	Malic Enzyme
MGTK	Methylglutaconase
MPC	Mitochondrial Pyruvate Carrier
mTHF	methyl-Tetrahydrofolate
OAA	Oxaloacetate
Oml	Oxomalonate
PCK	Phosphoenolpyruvate Carboxykinase
PCK	Pyruvate Carboxylase
PDHC	Pyruvate Dehydrogenase Complex
PEP	Phosphoenolpyruvate
PGM	Phosphoglucomutase
PGPase	2-phosphoglyceric acid Phosphatase
Phe	Phenylalanine
PHGDH	Phosphoglycerate Dehydrogenase
Php	Phosphohydroxypyruvate
PKM2	Pyruvate Kinase isoform M2
PL	Phospholipids
PPP	Pentose Phosphate Pathway
PSAT	Phosphoserine Aminotransferase
Pser	Phosphoserine
PSPH	Phosphoserine Phosphatase
pyr	Pyruvate
Q	Quinone
QH2	Quinol
SDH	Succinate Dehydrogenase
SDS	Serine Dehydratase
Ser	Serine
SHMT	Serine Hydroxymethyltransferase
SUCL	Succinate-CoA Ligase
THF	Tetrahydrofolate
Thr	Threonine
TR-Pase	Tartrate-resistant acid Phosphatase
Tyr	Tyrosine
Val	Valine

(2) Glc →→→ methylglyoxal →→→ pyruvate: This may occur through four different routes involving aldehyde dehydrogenase 9, zinc binding alcohol dehydrogenase domain containing two [more recently renamed to prostaglandin reductase 3 (Yu et al., 2013)] and at least two oxoaldehyde dehydrogenases; for details, see Vander Jagt and Hunsaker (2003). Methylglyoxal has been reported to trigger metastasis in breast, anaplastic thyroid, and colorectal cancer (Chiavarina et al., 2017; Antognelli et al., 2019; Nokin et al., 2019).

(3) Glc →→→ PEP → pyruvate: the terminal reaction is catalyzed by tartrate-resistant acid phosphatases (TRAP), the molecular identity of which remained unknown well after their biochemical characterization (Helwig et al., 1978; Chen and Chen, 1988; Hayman et al., 1989); they are most likely substantiated by a metalloprotein enzyme with the ability to catalyze the hydrolysis of orthophosphate monoesters under acidic conditions (Bull et al., 2002). The expression of this enzyme (TRAP) is a marker of bone disease in cancer patients (Nguyen et al., 1991; Koizumi and Ogata, 2002; Mose et al., 2003; Terpos et al., 2003; Chao et al., 2005).

(4) Glc →→→ PEP; PEP + GalNAc → GalNAc-1P + pyruvate: Terminal reaction catalyzed by N-acetylgalactosamine kinase isoforms 1 or 2 (Pastuszak et al., 1996). These enzymes are implicated in many signaling pathways inherent to carcinogenesis (Zeidan and Hart, 2010).

(5) Glc →→→ 3-PG → 2-PG (by phosphoglucomutase 1 or 2) → glycerate [probably through 2-phosphoglyceric acid phosphatase (Baranowski et al., 1968)] → 3-OH-pyr [by glyoxylate reductase (Mdluli et al., 2005)]; 3-OH-pyr + Ala (or glyoxylate) → Gly + pyruvate (or Ser): the terminal reaction is catalyzed by alanine-glyoxylate aminotransferase (Danpure et al., 2003). The mitochondrial isoform of the latter enzyme (alanine-glyoxylate aminotransferase isoform 2, AGXT2) has been reported to form glycine and pyruvate from alanine and glyoxylate; this reaction has been confirmed in normal tissues (Holmes and Assimos, 1998) and HepG2 cancer cells (Baker et al., 2004). The same reaction has been reported to take place in peroxisomes (Poore et al., 1997). On the other hand, loss of alanine-glyoxylate aminotransferase (AGXT) expression has been reported to accelerate the progression of hepatocellular carcinoma (Sun et al., 2019). A “futile cycle” may exist between 3-PG and glycerate through 2-phosphoglyceric acid phosphatase and glycerate kinase 1 and 2; glycerate kinase 2 is also found in the mitochondria (Guo et al., 2006).

(6) Glc →→→ 3-PG → phosphohydroxypyruvate (Php), catalyzed by phosphoglycerate dehydrogenase; Php + Ala → phosphoserine (Pser) + pyruvate, catalyzed by phosphoserine aminotransferase (PSAT) (Hirsch and Greenberg, 1967): PSAT overexpression is associated with increased tumorigenicity in human esophageal squamous cell carcinoma (Liu et al., 2016) and colon carcinomas (Yoon et al., 2015) and a poor outcome on tamoxifen therapy in recurrent breast cancer (De Marchi et al., 2017); conversely, its selective loss suppresses migration, invasion, and experimental metastasis in triple negative breast cancer (Metcalf et al., 2020).

(7) Glc →→→ 3-PG →Php (catalyzed by phosphoglycerate dehydrogenase); Php + Ala (or Glu) → Pser + pyruvate (or →Kg); the latter reaction is catalyzed by phosphoserine aminotransferase; Pser → Ser → pyruvate, catalyzed by serine dehydratase (Ogawa et al., 2006) or serine dehydratase-like (SDSL) (Ogawa et al., 2006). Notably, SDS was reported to be absent from human colon carcinomas (Snell et al., 1988).

(8) Glc →→→ Glyoxal →→→ glyoxylate (Lange et al., 2012); glyoxylate + 3-OH-pyr (or Ala) → Gly + pyruvate (or Ser): the terminal reaction is catalyzed by AGXT (for considerations related to cancer, see pathway no. 5).

(9) Glc →→→ 3-PG →Php (catalyzed by phosphoglycerate dehydrogenase); Php + Glu → Pser + →Kg; latter reaction catalyzed by phosphoserine aminotransferase; →Kg + Ala → Glu + pyruvate, catalyzed by alanine aminotransferase (GPT; for considerations related to cancer, see pathway no. 6).

Pathways Leading to Pyruvate Commencing From Glucose: Intermediates Transiting Through the Mitochondria

These pathways depend on one or more of three critical parameters: (1) glyoxylate entry into the mitochondria, (2) reversibility of the matrix phosphoenolpyruvate carboxykinase (PCK2), and (3) reversibility of the mitochondrial pyruvate carrier (MPC). Regarding glyoxylate, I was unable to find information on its transport across the inner mitochondrial membrane; however, it is known that it can be processed by the matrix-localized AGXT2 (Kakimoto et al., 1969). PCK2 expression and activity level are critical for many cancer types: in tumor-initiating enriched prostate cancer cell clones, PCK2 was overexpressed, and this correlated with more aggressive tumors and lower survival rates (Zhao et al., 2017); in lung cancer cell lines and in non-small cell lung cancer samples, PCK2 expression and activity were enhanced under low-glucose conditions (Leithner et al., 2015); finally, it was reported that PCK2 is required for glucose-independent cancer cell proliferation and tumor growth in vivo (Vincent et al., 2015). Regarding PCK2 reversibility, the enzyme has been shown to operate in the reaction toward OAA synthesis in mitochondria from rabbit liver (Carlsen et al., 1988), pigeon and rat liver (Wiese et al., 1996), guinea pig liver (Garber and Ballard, 1970; Garber and Salganicoff, 1973), rabbit enterocytes (Wuensch and Ray, 1997), chicken liver (Hebda and Nowak, 1982; Makinen and Nowak, 1983; Wilson et al., 1983; Erecinska and Wilson, 1984), and bullfrog liver (Goto et al., 1980). However, in Vincent et al. (2015), it was shown that a fraction of pyruvate originated from glutamine from PEP through PCK2. With respect to the reversibility of the MPC, this is a working hypothesis because there are no data showing pyruvate release from normally polarized mitochondria. Nevertheless, this is not a far-fetched hypothesis: succinate and other metabolites are effluxed from the mitochondria for non-metabolic roles against a hyperpolarized membrane potential (Mills et al., 2016), demonstrating that this is possible under appropriate conditions. It may be also relevant that pyruvate catabolism through the pyruvate dehydrogenase complex is associated with suppression of tumor growth in vitro and in vivo (Michelakis et al., 2008); relevant to this, genes coding for both the pyruvate dehydrogenase complex and pyruvate carboxylase in certain cancers are usually downregulated (Yuen et al., 2016); furthermore, pyruvate is found in blood plasma, urine, and cerebrospinal fluid, and its presence there is not associated with damage of plasma membranes. Of course, this does not mean that extracellular pyruvate originated from the mitochondria, but it indicates that it can cross the plasma membrane through monocarboxylate transporters, some of which are distributed both in plasma and in the inner mitochondrial membrane (Hussien and Brooks, 2011); indeed monocarboxylate transporter 1, which is one of the four known pyruvate transport mechanisms, was recently shown to export pyruvate from the cell (Hong et al., 2016); however, mitochondrial pyruvate export remains hypothetical especially in view of the fact that its exit is influenced by the membrane potential and →pH. It was also recently reported that loss of an MPC isoform prior to a tumorigenic stimulus doubled the frequency of adenoma formation and produced higher-grade tumors, and this was associated with a glycolytic metabolic phenotype and increased expression of stem cell markers (Bensard et al., 2020). Mindful of the above, these pathways are as shown in Figure 3 (yellow arrows).

FIGURE 3.

Pathways leading to pyruvate commencing from glucose, highlighted in yellow: intermediates transiting through the mitochondria. For abbreviations, see Table 1.

(10) Glc →→→ glyoxal →→→ glyoxylate: Glyoxylate enters the mitochondria; glyoxylate + Ala → Gly + pyruvate through AGXT2. Pyruvate may exit the mitochondria through the MPC (for considerations related to cancer, see pathway no. 5).

(11) Glc →→→ PEP which enters the mitochondria; PEP transport across the inner membrane of mammalian mitochondria has been demonstrated to occur by the tricarboxylate carrier by Robinson (1971) and the group of Soling et al. (1971) and Kleineke et al. (1973) and to a lesser extent by the adenine nucleotide carrier, shown by the Shug and Shrago (1973); Sul et al. (1976) and in Drahota et al. (1983) and reviewed in Passarella et al. (2003). The possibility of a PEP/pyruvate transporter has also been put forward (Satrustegui et al.,2007). More recently, PEP cycling via mitochondrial PEPCK evoking PEP transport across the inner mitochondrial membrane has also been demonstrated by the group of Kibbey (Stark et al., 2009); PEP → OAA by PCK2; OAA → pyruvate by reverse operation of PC. However, this is expected to be a very minor path. Pyruvate may exit the mitochondria through the MPC.

(12) Glc →→→ PEP; PEP enters the mitochondria through the means outlined in pathway 11. PEP → OAA by PCK2; OAA → pyruvate by FAHD1 (Pircher et al., 2011, 2015). FAHD1 also converts 3-acylpyruvate, acetylpyruvate, and fumarylpyruvate to pyruvate (Pircher et al., 2011). It is not known where acetylpyruvate comes from, but its existence is known since Krebs reported it (Krebs and Johnson, 1937). Pyruvate may exit the mitochondria through the MPC. FAHD1 depletion has been shown to induce premature senescence in human endothelial cells by inhibiting mitochondrial metabolism (Petit et al., 2017); however, this might be a double-edged sword since OXPHOS capacity has been inversely correlated with malignancy in several cell types (Zhou et al., 2003; Matoba et al., 2006; Hu et al., 2012; Hall et al., 2013; Bartesaghi et al., 2015; Nicolay et al., 2015; Capala et al., 2016; Smith et al., 2020).

(13) Glc →→→ PEP; PEP enters the mitochondria through the means outlined in pathway 11; PEP → OAA by PCK2; OAA → Mal by MDH2; Mal → pyruvate by ME2,3 (Zelewski and Swierczynski, 1991). Pyruvate may exit the mitochondria through the MPC. ME2 knockdown suppresses tumor growth in lung cancer (Ren et al., 2014), while ME2,3 deletions confer lethality in pancreatic cancer (Dey et al., 2017).

(14) Glc →→→ PEP; PEP enters the mitochondria through the means outlined in pathway 11; PEP → OAA by PCK2; OAA → Mal by MDH2; Mal exits the mitochondria; Mal → pyruvate by ME1 (Zelewski and Swierczynski, 1991; Loeber et al., 1994). ME1 knockdown inhibits the growth of colon cancer cells (Murai et al., 2017), and its overexpression is associated with larger breast tumor size, higher incidence of lymph node metastasis, and higher incidence of lymph–vascular invasion (Liu C. et al., 2020). In the same line, ME1 is associated with tumor budding—a phenomenon representing epithelial to mesenchymal transition—in oral squamous cell carcinomas (Nakashima et al., 2020).

(15) Glc →→→ PEP; PEP enters the mitochondria through the means outlined in pathway 11; PEP → OAA by PCK2; OAA + acetyl-CoA → citrate by CS; citrate exits the mitochondria through the dicarboxylate carrier; citrate + ATP + CoASH → acetyl-coA + ADP + Pi + OAA by ACLY (Chypre et al., 2012); OAA → Mal by MDH1; Mal → pyruvate by ME1 (for considerations related to cancer, see pathway no. 14).

(16) Glc →→→ PEP; PEP enters the mitochondria through the means outlined in pathway 11; PEP → OAA by PCK2; OAA + Glu →→Kg + Asp by GOT2; Asp exits the mitochondria; Asp + →Kg → Glu + OAA by GOT1; OAA → Mal by MDH1; Mal → pyruvate by ME1 (for considerations related to cancer, see pathway no. 14).

(17) Glc →→→ PEP; PEP enters the mitochondria through the means outlined in pathway 11; PEP → OAA by PCK2; OAA + acetyl-CoA → citrate by CS; citrate → cis-aconitate, intermediate of ACO2 reaction; cis-aconitate → itaconate by cADC; itaconate + CoASH + ATP (or GTP) → itaconyl-CoA + Pi + ADP (or GDP) by SUCL (Nemeth et al., 2016); itaconyl-CoA → citramalyl-CoA by methylglutaconase (MGTK); citramalyl-coA → acetyl-CoA + pyruvate by CLYBL (Shen et al., 2017). Pyruvate may exit the mitochondria through the MPC. CLYBL has been reported to be associated with colorectal cancer metastasis (Li and Peng, 2013). Furthermore, CLYBL was reported to be overexpressed in 465 out of 38,258 tumor samples in the COSMIC database⁸.

Pathways Leading to Pyruvate But Not Commencing From Glucose: Intermediates Not Transiting Through the Mitochondria

These pathways are shown in Figure 4 (green arrows).

FIGURE 4.

Pathways leading to pyruvate but not commencing from glucose, highlighted in green: intermediates not transiting through the mitochondria. For abbreviations, see Table 1.

(18) Ser → pyruvate, catalyzed by SDS or SDSL (for considerations related to cancer, see pathway no. 6).

(19) Ser →→→ PEP; PEP → pyruvate; terminal reaction catalyzed by tartrate-resistant acid phosphatase (TR-Pases; for considerations related to cancer, see pathway no. 3).

(20) Ser →→→ PEP; PEP + GalNAc → GalNAc-1P + pyruvate. The terminal reaction is catalyzed by N-acetylgalactosamine kinase isoforms 1 or 2 (for considerations related to cancer, see pathway no. 4).

(21) Ala → pyruvate, catalyzed by L-amino-acid oxidases (LAAO) (Nakano et al., 1967): Several mammalian LAAOs have been described, of which the enzyme “interleukin-4 induced gene 1” (IL4I1) is the best characterized (Castellano and Molinier-Frenkel, 2017); IL4I1 expression was reported to be associated with poor prognosis in human breast cancers (Finak et al., 2008).

(22) Ala + 2-oxoglrm → Gln + pyruvate, catalyzed by glutamine-pyruvate transaminase (GPAT) (Cooper and Meister, 1972; Cooper and Kuhara, 2014). GPAT is upregulated in many cancers in a MYC-dependent manner (Dong et al., 2020).

(23) Ala + 2-Oml → Aml + pyruvate, catalyzed by alanine-ketomalonate transaminase (ALXT) (Nagayama et al., 1958). I was unable to find relevant literature on ALXT expression or aminomalonate levels and cancer.

(24) Ala + αKg → Glu + pyruvate, catalyzed by GPT: GPT—similar to GPAT—is upregulated in many cancers in a MYC-dependent manner (Dong et al., 2020).

(25) Ala + OAA → Asp + pyruvate; enzyme unknown (Rowsell, 1956).

(26) Ala + Glyoxylate → Gly + pyruvate, catalyzed by alanine-glyoxylate aminotransferase (for considerations related to cancer, see pathway no. 5).

(27) Ala + 3-OH-pyr → Ser + pyruvate, catalyzed by alanine-glyoxylate aminotransferase (for considerations related to cancer, see pathway no. 5).

(28) Thr → Gly + acetaldehyde, catalyzed by SHMT1 (Garrow et al., 1993; Pinthong et al., 2014); Gly + 5,10 mTHF → THF + Ser, catalyzed by serine hydroxymethyltransferase 1; Ser → pyruvate, catalyzed by SDS or SDSL. SHMT1 knockdown induces apoptosis in lung cancer cells (Paone et al., 2014), and SHMT inhibitors block the growth of many human cancer cells (Ducker et al., 2017). Patients with high SHMT2 expression exhibit a shorter overall survival rate compared with patients with low expression (Koseki et al., 2018; for further considerations related to SDS or SDSL and cancer, see pathway no. 6).

(29) Asp + αKg → Glu + OAA, catalyzed by GOT1; OAA → Mal by MDH1; Mal → pyruvate by ME1 (for considerations related to cancer, see pathway no. 14).

(30) 4-OH-proline →→→ pyruvate, through glyoxylate formation (see pathway no. 26).

(31) Cys →→→ pyruvate through the sulfinate pathway (Stipanuk, 1979, 2020). Notably, in pancreatic cancer cells exhibiting PKM1/2 knockdown, 20% of intracellular pyruvate originated from cysteine (Yu et al., 2019). The contribution of cysteine catabolism to cancer has been extensively reviewed by Serpa (2020).

(32) Cys → 3-sulfino-L-alanine catalyzed by aspartate 4-decarboxylase (Liu et al., 2012); 3-sulfino-L-alanine is transaminated to 3-sulfinopyruvate by either aspartate aminotransferase or deaminated to the same product by cysteine sulfinic acid deaminase; 3-sulfinopyruvate is non-enzymatically converted to sulfite and pyruvate (Stipanuk, 2020; for considerations related to cancer, see pathway no. 31).

(33) Cys →→→ H₂S + pyruvate through the 3-mercaptopyruvate pathway (Nagahara and Sawada, 2006). Cys can also transaminate with →-ketoglutarate to form glutamate and 3-mercaptopyruvate though GOT1, exhibiting cysteine transaminase activity. The catabolism of 3-mercaptopyruvate toward pyruvate is outlined in the reactions below (pathway no. 34; for considerations related to cancer, see pathway no. 31).

(34) L-cysteine is isomerized to D-cysteine by cysteine racemase (2-amino-3-mercaptopropionic acid racemase) (Soda and Osumi, 1969); D-Cys is converted to 3-mercaptopyruvate by D-amino acid oxidase and, in turn, to pyruvate and H₂S by 3-mercaptopyruvate sulfurtransferase (3MST) (Shibuya et al., 2013) or thiosulfate sulfurtransferase (TST) (Pallini et al., 1991). The possibility of conversion of D-Cys to pyruvate by D-cysteine desulfhydrase (Nagasawa et al., 1985) in mammalian cells is yet to be reported. 3-Mercaptopyruvate can also react with hydrogen cyanide, forming pyruvate and thiocyanate in a reaction catalyzed by 3MST or TST; obviously, this is only a very minor route of pyruvate production due to cyanide toxicity (Bhandari et al., 2014; for further considerations related to cancer, see pathway no. 31).

(35) Ser → dehydroalanine (2-aminoacrylate) by serine dehydratase (SDS), serine dehydratase-like protein (SDSL), or serine racemase (SRR): Dehydroalanine can further hydrolyze to NH₃ and pyruvate through SDS, SDSL, or SRR (Kashii et al., 2005); sometimes this reaction is referred to as hydrolysis by “2-aminoacrylate aminohydrolase.” Dehydroalanine can also spontaneously hydrolyze to NH₃ and pyruvate through the intermediate 2-iminopropanoate; the latter later part of this spontaneous hydrolysis can be accelerated by 2-iminopropanoate deaminase (Lambrecht et al., 2012). Dehydroalanine can also be derived from 2 3,5-diiodo-L-tyrosine or 3,5-diiodo-L-tyrosine by thyroid peroxidase in the process of forming thyroxine and triiodothyronine, respectively (Gavaret et al., 1980). The crucial importance of serine metabolism for the growth and survival of proliferating cells is extensively reviewed in Yang and Vousden (2016) and Newman and Maddocks (2017).

(36) Se-methyl-L-selenocysteine (SeMSC, Se-methylselenocysteine, methyl selenocysteine) can be deaminated to methaneselenol, NH₃, and pyruvate by selenocysteine lyase (Esaki et al., 1982). SeMSC can be found in many edible plants, including garlic, onions, and broccoli, as well as in dietary supplements (Yang and Jia, 2014). SeMSC was shown to exhibit anticarcinogenic properties (Ip et al., 1991; Medina et al., 2001) and even potentiate the antitumor activity of anticancer drugs (Cao et al., 2014).

(37) Val →→→ 2-methyl-3-oxopropanoate; 2-methyl-3-oxopropanoate can get transaminated with alanine by AGXT2 to D-3-amino-isobutanoate + pyruvate (Kakimoto et al., 1969). The overexpression of enzymes participating in valine catabolism is associated with poor prognosis in prostate cancer (Mayers et al., 2016) and tumors of the colon (Shan et al., 2019). The role of valine in cancer has been extensively reviewed in Ananieva and Wilkinson (2018) and Lieu et al. (2020).

(38) Leu →→→ 3-methylbutanoyl-CoA; the latter compound is converted to isobutyryl-CoA through branched-chain fatty acid metabolism (many steps); isobutyryl-CoA →→→ 2-methyl-3-oxopropanoate; 2-methyl-3-oxopropanoate can get transaminated with alanine by AGXT2 to D-3-amino-isobutanoate + pyruvate (Kakimoto et al., 1969). Because leucine catabolism shares many steps with that of valine, for considerations related to cancer, see pathway no. 37.

(39) Ile →→→ 2-methylbutanoyl-CoA; the latter compound is converted to isobutyryl-CoA through branched-chain fatty acid metabolism (many steps); isobutyryl-CoA →→→ 2-methyl-3-oxopropanoate; 2-methyl-3-oxopropanoate can get transaminated with alanine by AGXT2 to D-3-amino-isobutanoate + pyruvate (Kakimoto et al., 1969). Because isoleucine catabolism shares many steps with that for valine, for considerations related to cancer, see pathway no. 37.

(40) Pro + αKg + O₂ → CO₂ + succinate + trans-4-hydroxy-L-proline, catalyzed by prolyl 4-hydroxylase subunit alpha (isoforms 1, 2, or 3); trans-4-hydroxy-L-proline is then converted to L-1-pyrroline-3-hydroxy-5-carboxylate, also yielding NAD(P)H, by either pyrroline-5-carboxylate reductase (isoforms 1, 2, or 3) or left–right determination factor 1 (LEFTY1), a member of the TGF-→ family of proteins; L-1-pyrroline-3-hydroxy-5-carboxylate can be converted to L-erythro-4-hydroxyglutamate, also yielding NAD(P)H, by aldehyde dehydrogenase 4 family member A1; in turn, L-erythro-4-hydroxyglutamate is transaminated with either OAA by GOT2, yielding 4-hydroxy-2-oxoglutarate + aspartate, or →Kg by GOT1 or GOT2, yielding 4-hydroxy-2-oxoglutarate + glutamate; finally, 4-hydroxy-2-oxoglutarate is converted to glyoxylate and pyruvate by 4-hydroxy-2-oxoglutarate glyoxylate-lyase. It is relevant that increased proline catabolism has been recently reported to support metastasis (Elia et al., 2017). Arg, through either interconversion to metabolites as for proline catabolism or through citrulline/ornithine and the fumarate nucleotide cycle will also lead to pyruvate formation; however, this probably requires inter-organ communication and, thus, may not be found within a single cell. The crucial role of proline catabolism in tumor growth and metastatic progression is extensively reviewed in Phang (2019) and D’Aniello et al. (2020).

Pathways Leading to Pyruvate But Not Commencing From Glucose: Intermediates Transiting Through the Mitochondria

These pathways are shown in Figure 5 (blue arrows).

FIGURE 5.

Pathways leading to pyruvate but not commencing from glucose, highlighted in blue: intermediates transiting through the mitochondria. For abbreviations, see Table 1.

(41) Thr →→→ acetyl-CoA; acetyl-CoA + OAA → citrate, catalyzed by CS; citrate exits the mitochondria through the dicarboxylate carrier; citrate + ATP + CoASH → Acetyl-coA + ADP + Pi + OAA by ACLY (Chypre et al., 2012); OAA → Mal by MDH1; Mal → pyruvate by ME1. The potential role of threonine catabolism in cancer is reviewed in Tsun and Possemato (2015) and Lieu et al. (2020) (for further considerations regarding ME1 and cancer, see pathway no. 14).

(42) Thr →→→ acetyl-CoA; acetyl-CoA + OAA → citrate, catalyzed by CS; citrate → cis-aconitate, intermediate of ACO2 reaction; cis-aconitate → itaconate by cADC; itaconate + CoASH + ATP (or GTP) → itaconyl-CoA + Pi + ADP (or GDP) by SUCL; itaconyl-CoA → citramalyl-CoA by MGTK; citramalyl-coA → acetyl-CoA + pyruvate by CLYBL. Pyruvate may exit the mitochondria through the MPC (regarding threonine and cancer, see pathway no. 41; regarding CLYBL and cancer, see pathway no. 17).

(43) Asn →→→ Asp; Asp + αKg → Glu + OAA by GOT2; OAA by PCK2; OAA → pyruvate by reverse operation of PC. However, this is expected to be a path of a very minor flux. Pyruvate may exit the mitochondria through the MPC. The crucial role of asparagine availability in cancer is explored in Panosyan et al. (2014); Krall et al. (2016), and Knott et al. (2018). However, more emphasis on asparagine availability for anabolic, rather than catabolic, purposes is given.

(44) Asn →→→ Asp; Asp + αKg → Glu + OAA by GOT2; OAA → pyruvate by acylpyruvase (FAHD1). Pyruvate may exit the mitochondria through the MPC (for considerations related to cancer, see pathways no. 12 and 37).

(45) Asn →→→ Asp; Asp + αKg → Glu + OAA by GOT2; OAA → Mal by MDH2; Mal → pyruvate by ME2,3. Pyruvate may exit the mitochondria through the MPC (for considerations related to cancer, see pathways no. 13 and 37).

(46) Asn →→→ Asp; Asp + αKg → Glu + OAA by GOT2; OAA → Mal by MDH2; Mal exits the mitochondria; Mal → pyruvate by ME1 (for considerations related to cancer, see pathways no. 14 and 37).

(47) Tyr, Phe →→→ Fum; Fum → Mal by FH; Mal → pyruvate by ME2,3 (for considerations related to cancer, see pathway no. 13).

(48) Tyr, Phe →→→ Fum; Fum → Mal by FH; Mal exits the mitochondria; Mal → pyruvate by ME1 (for considerations related to cancer, see pathway no. 14).

(49) Tyr, Phe →→→ Fum; Fum → Mal by FH; Mal → OAA by MDH2; OAA → pyruvate by acylpyruvase (FAHD1). Pyruvate may exit the mitochondria through the MPC (for considerations related to cancer, see pathway no. 12).

(50) Thr →→→ acetyl-CoA; acetyl-CoA + OAA → citrate, catalyzed by CS; citrate exits the mitochondria through the dicarboxylate carrier; citrate + ATP + CoASH → acetyl-coA + ADP + Pi + OAA by ACLY; OAA → PEP by PCK1; PEP enters the mitochondria; PEP → OAA by PCK2; OAA → pyruvate by acylpyruvase (FAHD1). Pyruvate may exit the mitochondria through the MPC (for considerations related to cancer, see pathway no. 12).

(51) Thr →→→ acetyl-CoA; acetyl-CoA + OAA → citrate, catalyzed by CS; citrate exits the mitochondria through the dicarboxylate carrier; citrate + ATP + CoASH → acetyl-coA + ADP + Pi + OAA by ACLY; OAA → PEP by PCK1; PEP enters mitochondria; PEP → OAA by PCK2; OAA → Mal by MDH2; Mal → pyruvate by ME2,3. Pyruvate may exit the mitochondria through the MPC (for considerations related to cancer, see pathway no. 13).

(52) Thr →→→ acetyl-CoA; acetyl-CoA + OAA → citrate, catalyzed by CS; citrate exits the mitochondria through the dicarboxylate carrier; citrate + ATP + CoASH → acetyl-CoA + ADP + Pi + OAA by ACLY; OAA → PEP by PCK1; PEP enters the mitochondria; PEP → OAA by PCK2; OAA → Mal by MDH2; Mal exits the mitochondria; Mal → pyruvate by ME1 (for considerations related to cancer, see pathway no. 14).

(53) Thr →→→ acetyl-CoA; acetyl-CoA + OAA → citrate, catalyzed by CS; citrate exits the mitochondria through the dicarboxylate carrier; citrate + ATP + CoASH → Acetyl-coA + ADP + Pi + OAA by ACLY; OAA → PEP by PCK1; PEP + GalNAc → GalNAc-1P + pyruvate. Terminal reaction catalyzed by N-acetylgalactosamine kinase isoforms 1 or 2 (for considerations related to cancer, see pathway no. 4).

(54) Thr →→→ acetyl-CoA; acetyl-CoA + OAA → citrate, catalyzed by CS; citrate exits the mitochondria through the dicarboxylate carrier; citrate + ATP + CoASH → acetyl-coA + ADP + Pi + OAA by ACLY; OAA → PEP by PCK1; PEP → pyruvate; the terminal reaction is catalyzed by tartrate-resistant acid phosphatases (for considerations related to cancer, see pathway no. 3).

Incompletely Characterized Reactions Forming Pyruvate

In the literature, some reactions have been described to produce pyruvate but are incompletely characterized. These are collectively listed below:

(55) O-carbamoyl-L-serine + H₂O → pyruvate + 2 NH₃, catalyzed by carbamoyl-serine ammonia lyase (Copper and Meister, 1973). O-Carbamoyl-L-serine is a weak inhibitor of a phosphate-dependent glutaminase (Shapiro et al., 1979); mindful of the crucial importance of glutamine catabolism through glutaminases in many cancer types, this route of pyruvate provision is probably minor.

(56) L-Cysteine-S-conjugate + H₂O → a thiol + NH₃ + pyruvate, catalyzed by cysteine S-conjugate →-lyases (Cooper and Pinto, 2006). The possibility of cysteine S-conjugate β-lyases metabolizing anticancer agents is reviewed in Cooper et al. (2011).

(57) cystathionine + H₂O → L-homocysteine + pyruvate + NH₃ or cysteine + H₂O → sulfide + NH₃ + pyruvate or cystine → thiocysteine + pyruvate + NH₃, all catalyzed by cystathionine gamma-lyase (Stipanuk et al., 2006; Chiku et al., 2009). Cystathionine gamma-lyase was reported to be upregulated in bone−metastatic PC3 cells, and its knockdown suppressed tumor growth and metastasis (Wang et al., 2019). In the same line, this enzyme was shown to be upregulated and played a crucial role in the proliferation and migration of breast cancer cells (You et al., 2017).

(58) L-Serine O-sulfate + H₂O → pyruvate + NH₃ + sulfate catalyzed by serine-sulfate ammonia-lyase (Tudball and Thomas, 1972). I was unable to find relevant literature on serine-sulfate ammonia-lyase expression or L-serine O-sulfate levels and cancer.

(59) N-Acetylneuraminate → N-acetyl-D-mannosamine + pyruvate catalyzed by N-acetylneuraminate lyase (Brunetti et al., 1962); relevant to this, treatment of HL-60 cells by phorbol esters leads to a marked increase in the activity of this enzyme (Warren, 1986).

(60) D-Alanine + H₂O + O₂ → pyruvate + NH₃ + H₂O₂ catalyzed by DAAO (Nagata et al., 1992; Abe et al., 2005; Fuchs et al., 2005; Smith et al., 2009). The interaction of D-alanine (and other D-amino acids) with tumors is reviewed in Bastings et al. (2019).

(61) L-Alanine → pyruvate + NH₃ catalyzed by glutamate dehydrogenase; this reaction exhibits a weak activity (Silverstein, 1974). The role of glutamate dehydrogenase in cancer cells has been extensively reviewed in Moreno-Sanchez et al. (2020).

(62) 2-Oxosuccinamic acid + Ala → Asn + pyruvate, catalyzed by asparagine aminotransferase (Cooper, 1977; Maul and Schuster, 1986). The origin of 2-oxosuccinamic acid is not known (Cooper et al., 1987). I was unable to find relevant literature on 2-oxosuccinamic acid levels and cancer.

(63) Pyruvate oxime + acetone → pyruvate + acetone oxime, catalyzed by oximinotransferase (Omura et al., 1956). Due to acetone volatility, this is probably a very minor pathway for pyruvate production.

(64) Methylmalonyl-CoA + pyruvate → propionyl-CoA + oxaloacetate catalyzed by methylmalonyl-CoA carboxytransferase (Swick and Wood, 1960). This reaction is reversible and thus may yield pyruvate. I was unable to find relevant literature on methylmalonyl-CoA carboxytransferase and cancer.

(65) L-Alanine + 3-oxopropanoate → pyruvate + →-alanine, catalyzed by either →-alanine-pyruvate transaminase (Ito et al., 2001) or alanine-glyoxylate aminotransferase isoform 2 (Lee et al., 1995) (for considerations related to cancer, see pathway no. 5).

(66) Phenylpyruvate + L-alanine → L-phenylalanine + pyruvate catalyzed by phenylalanine (histidine) transaminase (Minatogawa et al., 1977). Phenylpyruvate has been reported to inhibit pyruvate kinase activity in human brain (Weber, 1969), thus enhancing PK-bypassing pathways. Phenylpyruvate levels were also found to be increased in ovarian cancers (Fong et al., 2011).

(67) 2-Oxoisohexanoate + L-alanine → L-leucine + pyruvate, catalyzed by the mitochondrial branched-chain L-amino acid aminotransferase (Schadewaldt et al., 1995). The role of branched-chain L-amino acid aminotransferase in cancer has been reviewed in Ananieva and Wilkinson (2018).

(68) PCK1, ME1, and ME2,3 may also convert OAA to CO₂ and pyruvate (Sauer, 1973; Carlson et al., 1978; Bukato et al., 1995; Lee et al., 1995) (for considerations related to cancer, see pathway nos. 13 and 14).

(69) Salsolinol can be converted to salsolinol-1-carboxylate by salsolinol synthetase which can then be catabolized to dopamine and pyruvate (by an unknown enzyme); salsolinol is an endogenous catechol isoquinoline detected in humans derived from dopamine metabolism (Sandler et al., 1973; Collins et al., 1979). Salsolinol has been implicated in the initiation and promotion of alcohol-related breast carcinogenesis (Murata et al., 2016).

Pathways Leading to L-Lactate and D-Lactate Including Those Not Going Through Lactate Dehydrogenase

These pathways are shown in Figure 6 (brown arrows).

FIGURE 6.

Pathways leading to L- and D-lactate, including those not going through lactate dehydrogenase, highlighted in brown. For abbreviations, see Table 1.

Lactate—unlike pyruvate—exhibits chirality; thus, it exists in L- or D- configuration. In humans, a putative D-lactate dehydrogenase is known to exist (Flick and Konieczny, 2002; Ewaschuk et al., 2005; Chen et al., 2015). In metabolomics experiments, it is uncommon to distinguish between L- and D-lactate even although it is possible by using special columns. In this section, D- and L-lactate-forming pathways are outlined, including those not going through LDH:

(70) D-lactate formation by methylglyoxal and intestinal flora (Chen et al., 2015) (for considerations related to cancer, see pathway no. 2).

(71) Pyruvate + QH₂ → D-lactate + Q, catalyzed by D2HGDH in the mitochondrial matrix (Cammack, 1969, 1970). Mutations in D2HGDH have been reported to be involved in multiple types of cancers but render the enzyme hypoactive or inert (Ye et al., 2018); thus, it is unlikely for this route to be important regarding pyruvate production.

(72) D- (or L-) Lactate + 2 ferricytochrome → 2 ferrocytochrome C + 2 H⁺ + pyruvate, catalyzed by D-lactate dehydrogenase; this reaction is mentioned in several databases, but no reference is given.

(73) D- (or L-) Lactate + 2 ferricytochrome → 2 ferrocytochrome C + 2 H⁺ + pyruvate, catalyzed by cytochrome B5 domain-containing protein 1; this reaction is mentioned in several databases, but no reference is given.

(74) Pyruvate + NADPH → NADP⁺ + L-lactate, catalyzed by ADH (Bosron and Prairie, 1972). The many roles of ADH in malignant neoplasms have been extensively reviewed in Orywal and Szmitkowski (2017).

(75) Pyruvate + H₂O₂ → L-lactate + O₂, catalyzed by hydroxyacid oxidases (HAO1,2,3) (Fry and Richardson, 1979; Vignaud et al., 2007). However, in Jones et al. (2000), no HAO activity was reported. In primary pancreatic tumors, HAO3 is strongly downregulated (Thakur et al., 2008). HAO2 was reported to inhibit the malignancy of clear cell renal cell carcinoma cells. Overall, it is unlikely for this to be a substantial pathway in yielding pyruvate in cancer.

(76) Protein deglycase (E.C. 3.5.1.124) may form D-lactate from proteins (Richarme et al., 2015; Richarme and Dairou, 2017). Relevant to this, the deglycase DJ-1/Park7 is important for cancer cell survival (Vasseur et al., 2009).

(77) Methylglyoxal spontaneously forms a hemithioacetal adduct with GSH; subsequently, glyoxalase I (lactoylglutathione lyase; EC 4.4.1.5) produces S-D-lactoylglutathione from this adduct (Thornalley, 1990), and glyoxalase II (hydroxyacylglutathione hydrolase; EC 3.1.2.6), in turn, hydrolyzes S-D-lactoylglutathione to D-lactate + GSH (Cordell et al., 2004) (for considerations related to cancer, see pathway no. 2).

Finally, it is worth mentioning that LDH may process substrates other than pyruvate and lactate, interconverting glyoxylate + NAD⁺ to oxalate + NADH or α-ketobutyrate to →-hydroxybutyrate or L-glycerate to hydroxypyruvate (Dawkins and Dickens, 1965; Kim and Whitesides, 1988).

Pathways Leading to Pyruvate Commencing From Glutamine (Glutaminolysis)

It is a well-known fact that most cancer cells grow much better when feeding media contain glutamine; this spurred from the pioneering studies of Eagle et al. (1956), showing the dependence of cancer cells growing in monolayer cultures on glutamine. The many critical roles of glutamine in tumor metabolism is reviewed in Altman et al. (2016). From the energetic point of view it were Reitzer et al. (1979) who first showed that glutamine, not sugars, is the main energy source in cultured HeLa cells and that carbon atoms from glutamine incorporate into lactate, but not more than 13%. Zielke et al. (1980), likewise reported that human diploid fibroblasts metabolize up to 13% of media glutamine to lactate. In the same line of thought, Scott et al. (2011), showed that, in human melanoma cell lines, glutamine did not significantly label lactate, in agreement with the data of Ta and Seyfried (2015) reporting that, in a murine glioblastoma cell line, minimal amounts of lactate derived from glutamine were detected. Le et al. (2012), as well as Son et al. (2013) likewise showed that ¹³C-labeled atoms in glutamine appear in lactate also to a minimal extent. However, in a study published by DeBerardinis et al. (2007), ∼60% of the glutamine metabolized by SF188 cells was claimed to be converted to lactate, although they seemed to combine this percentage with that of alanine production. The pathway of converting glutamine to pyruvate (and lactate), referred to by McKeehan (1982) as “glutaminolysis,” has been considered a hallmark of tumor metabolism; however, this is a misconception: in normal tissues, ∼18% of glutamine carbons appear in lactate (Windmueller and Spaeth, 1974), as opposed to ∼10–13% (or less) in tumor cells (see the references above). Thus, if anything, cancer cells exhibit a decrease in glutamine-to-lactate conversion exactly as anticipated, mindful that glutamine provides both energy and building blocks for several biosynthetic processes of cancer. Although glutaminolysis was originally attributed to the pathway Gln → Glu → aKg → succinyl-CoA → succinate → fumarate → malate (exiting the mitochondria) → pyruvate (through malic enzyme), several other routes may also contribute (outlined below; see Figure 7).

FIGURE 7.

Pathways leading to pyruvate commencing from glutamine (glutaminolysis), highlighted in red. For abbreviations, see Table 1.

(78) (For the sake of completion, the glutaminolysis pathway proposed by McKeehan (1982) is repeated in the present entry) Gln → Glu → aKg → succinyl-CoA → succinate → fumarate → malate; malate exits the mitochondria → pyruvate; this last step is catalyzed by cytosolic malic enzyme (ME1).

(79) Gln → Glu → aKg → isocitrate → cis-aconitate → itaconate by cADC; itaconate + CoASH + ATP (or GTP) → itaconyl-CoA + Pi + ADP (or GDP) by SUCL (Nemeth et al., 2016); itaconyl-CoA → citramalyl-CoA by methylglutaconase (MGTK); citramalyl-coA → acetyl-CoA + pyruvate by CLYBL (Shen et al., 2017). Pyruvate may exit the mitochondria through the MPC.

(80) Gln → Glu → aKg → isocitrate → cis-aconitate → citrate, exiting the mitochondria → citrate + ATP + CoASH → acetyl-coA + ADP + Pi + OAA by ACLY (Chypre et al., 2012); OAA → Mal by MDH1; Mal → pyruvate by ME1.

(81) Gln → Glu → aKg → isocitrate → cis-aconitate → citrate, exiting the mitochondria → citrate + ATP + CoASH → acetyl-coA + ADP + Pi + OAA by ACLY; OAA → PEP by PCK1; PEP + GalNAc → GalNAc-1P + pyruvate. The terminal reaction is catalyzed by N-acetylgalactosamine kinase isoforms 1 or 2.

(82) Gln → Glu → aKg → isocitrate → cis-aconitate → citrate, exiting the mitochondria → citrate + ATP + CoASH → acetyl-coA + ADP + Pi + OAA by ACLY; OAA → PEP by PCK1; PEP → pyruvate; the terminal reaction is catalyzed by tartrate-resistant acid phosphatases.

(83) Gln → Glu → aKg → succinyl-CoA → succinate → fumarate → malate → pyruvate by ME2,3; pyruvate may exit the mitochondria through the MPC.

(84) Gln → Glu → aKg; aKg transaminates with Asp forming Glu and OAA, by GOT2; OAA → pyruvate by FAHD1 (Pircher et al., 2011, 2015); pyruvate may exit the mitochondria through the MPC.

(85) Gln → Glu → aKg; aKg transaminates with Asp forming Glu and OAA, by GOT2; OAA → Mal by MDH2; Mal exits the mitochondria; Mal → pyruvate by ME1 (Zelewski and Swierczynski, 1991; Loeber et al., 1994).

(86) Gln → Glu → aKg; aKg transaminates with Asp forming Glu and OAA, by GOT2; OAA → Mal by MDH2; malate → pyruvate by ME2,3; pyruvate may exit the mitochondria through the MPC.

Energetics of Glycolysis With Kinetically Inactive PK

Glycolysis yields a net of two ATP molecules per glucose molecule; however, in view of an inactive PK while pyruvate is made through PK-bypass pathways, net ATP production from glycolysis is expected to be zero. Although the importance of high-energy phosphate generation has been downplayed in cancer tissues (Vander Heiden et al., 2009), it cannot be ignored that—according to the BRENDA database—among the 336 enzymatic reactions requiring ATP in a cell (without even considering quantitatively important, non-enzymatic mechanisms such as Na⁺/K⁺ ATPase), 125 of them occur in the cytosol. Clearly, while it is imperative to prevent phosphofructokinase and hexokinase from ATP-dependent feedback inhibition and allow a high flux of glycolysis for the sake of generating intermediates shuttled toward other pathways, ATP is still needed for many other reactions. Crunching the numbers regarding cytosolic energetics is a daunting task, but what is definite is that a cell with nearly zero ATP production from glycolysis may not harbor ATP-consuming mitochondria, for whatever reason (hypoxia, mtDNA mutations, etc.). This can be solved by maintaining the adenine nucleotide translocase in “forward” mode, i.e., providing ATP to the cytosol which is made by SUCL supported by glutaminolysis (Chinopoulos et al., 2010). Production of pyruvate and, therefore lactate is still maintained by the PK-bypassing pathways so as to thwart a reductive stress as pyruvate-to-lactate by LDH maintains a low NADH/NAD⁺ ratio. Finally, it is important to emphasize that this lack of ATP generation by glycolysis due to PK inhibition does not only occur in neoplastic tissues, but it seems to be a more general pathophysiological mechanism also present in tissue ischemia: it was recently reported that during acute kidney injury, PK was inhibited by oxidative/nitrosative stress for the purpose of diverting glycolytic intermediates toward the pentose phosphate pathway which, in turn, yielded reducing equivalents and mounted a better response during the reperfusion phase where ROS are formed, thus increasing the chances for organ survival (Zhou et al., 2019).

Conclusion

The above considerations aim to (i) highlight that L-lactate can still be produced from pyruvate using carbon atoms originating from glucose or other substrates in cells with kinetically impaired pyruvate kinase and (ii) show that the mitochondria may contribute to cancer metabolism irrespective of oxidative phosphorylation by providing means of contributing to pyruvate production. Having said that, it is important to emphasize that none of the aforementioned reactions take into account the potential regulatory effects of metabolites on other reactions such as those occurring on PK by amino acids (Chaneton et al., 2012; Yuan et al., 2018). In addition, each enzyme probably exhibits different kinetic and thermodynamic constraints which control the overall flux, which also means that many of these pathways may not operate simultaneously. Such exponentially increasing complexity of a system precludes the possibility of predictions and modeling, though I would be happy to be proven wrong.

Author Contributions

CC wrote and edited the manuscript.

Conflict of Interest

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.