Showing papers on "Pyruvate kinase published in 2019"

Metabolic reprogramming by the S -nitroso-CoA reductase system protects against kidney injury

Abstract: Endothelial nitric oxide synthase (eNOS) is protective against kidney injury, but the molecular mechanisms of this protection are poorly understood1,2. Nitric oxide-based cellular signalling is generally mediated by protein S-nitrosylation, the oxidative modification of Cys residues to form S-nitrosothiols (SNOs). S-nitrosylation regulates proteins in all functional classes, and is controlled by enzymatic machinery that includes S-nitrosylases and denitrosylases, which add and remove SNO from proteins, respectively3,4. In Saccharomyces cerevisiae, the classic metabolic intermediate co-enzyme A (CoA) serves as an endogenous source of SNOs through its conjugation with nitric oxide to form S-nitroso-CoA (SNO-CoA), and S-nitrosylation of proteins by SNO-CoA is governed by its cognate denitrosylase, SNO-CoA reductase (SCoR)5. Mammals possess a functional homologue of yeast SCoR, an aldo-keto reductase family member (AKR1A1)5 with an unknown physiological role. Here we report that the SNO-CoA-AKR1A1 system is highly expressed in renal proximal tubules, where it transduces the activity of eNOS in reprogramming intermediary metabolism, thereby protecting kidneys against acute kidney injury. Specifically, deletion of Akr1a1 in mice to reduce SCoR activity increased protein S-nitrosylation, protected against acute kidney injury and improved survival, whereas this protection was lost when Enos (also known as Nos3) was also deleted. Metabolic profiling coupled with unbiased mass spectrometry-based SNO-protein identification revealed that protection by the SNO-CoA-SCoR system is mediated by inhibitory S-nitrosylation of pyruvate kinase M2 (PKM2) through a novel locus of regulation, thereby balancing fuel utilization (through glycolysis) with redox protection (through the pentose phosphate shunt). Targeted deletion of PKM2 from mouse proximal tubules recapitulated precisely the protective and mechanistic effects of S-nitrosylation in Akr1a1-/- mice, whereas Cys-mutant PKM2, which is refractory to S-nitrosylation, negated SNO-CoA bioactivity. Our results identify a physiological function of the SNO-CoA-SCoR system in mammals, describe new regulation of renal metabolism and of PKM2 in differentiated tissues, and offer a novel perspective on kidney injury with therapeutic implications.

The HGF-MET axis coordinates liver cancer metabolism and autophagy for chemotherapeutic resistance

Abstract: Notwithstanding the numerous drugs available for liver cancer, emerging evidence suggests that chemotherapeutic resistance is a significant issue. HGF and its receptor MET play critical roles in liver carcinogenesis and metastasis, mainly dependent on the activity of receptor tyrosine kinase. However, for unknown reasons, all HGF-MET kinase activity-targeted drugs have failed or have been suspended in clinical trials thus far. Macroautophagy/autophagy is a protective 'self-eating' process for resisting metabolic stress by recycling obsolete components, whereas the impact of autophagy-mediated reprogrammed metabolism on therapeutic resistance is largely unclear, especially in liver cancer. In the present study, we first observed that HGF stimulus facilitated the Warburg effect and glutaminolysis to promote biogenesis in multiple liver cancer cells. We then identified the pyruvate dehydrogenase complex (PDHC) and GLS/GLS1 as crucial substrates of HGF-activated MET kinase; MET-mediated phosphorylation inhibits PDHC activity but activates GLS to promote cancer cell metabolism and biogenesis. We further found that the key residues of kinase activity in MET (Y1234/1235) also constitute a conserved LC3-interacting region motif (Y1234-Y1235-x-V1237). Therefore, on inhibiting HGF-mediated MET kinase activation, Y1234/1235-dephosphorylated MET induced autophagy to maintain biogenesis for cancer cell survival. Moreover, we verified that Y1234/1235-dephosphorylated MET correlated with autophagy in clinical liver cancer. Finally, a combination of MET inhibitor and autophagy suppressor significantly improved the therapeutic efficiency of liver cancer in vitro and in mice. Together, our findings reveal an HGF-MET axis-coordinated functional interaction between tyrosine kinase signaling and autophagy, and establish a MET-autophagy double-targeted strategy to overcome chemotherapeutic resistance in liver cancer. Abbreviations: ALDO: aldolase, fructose-bisphosphate; CQ: chloroquine; DLAT/PDCE2: dihydrolipoamide S-acetyltransferase; EMT: epithelial-mesenchymal transition; ENO: enolase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GLS/GLS1: glutaminase; GLUL/GS: glutamine-ammonia ligase; GPI/PGI: glucose-6-phosphate isomerase; HCC: hepatocellular carcinoma; HGF: hepatocyte growth factor; HK: hexokinase; LDH: lactate dehydrogenase; LIHC: liver hepatocellular carcinoma; LIR: LC3-interacting region; PDH: pyruvate dehydrogenase; PDHA1: pyruvate dehydrogenase E1 alpha 1 subunit; PDHX: pyruvate dehydrogenase complex component X; PFK: phosphofructokinase; PK: pyruvate kinase; RTK: receptor tyrosine kinase; TCGA: The Cancer Genome Atlas.

Safety and efficacy of mitapivat in pyruvate kinase deficiency

Abstract: Background Pyruvate kinase deficiency is caused by mutations in PKLR and leads to congenital hemolytic anemia. Mitapivat is an oral, small-molecule allosteric activator of pyruvate kinase ...

FOXK1 and FOXK2 regulate aerobic glycolysis.

Abstract: Adaptation to the environment and extraction of energy are essential for survival. Some species have found niches and specialized in using a particular source of energy, whereas others-including humans and several other mammals-have developed a high degree of flexibility1. A lot is known about the general metabolic fates of different substrates but we still lack a detailed mechanistic understanding of how cells adapt in their use of basic nutrients2. Here we show that the closely related fasting/starvation-induced forkhead transcription factors FOXK1 and FOXK2 induce aerobic glycolysis by upregulating the enzymatic machinery required for this (for example, hexokinase-2, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase), while at the same time suppressing further oxidation of pyruvate in the mitochondria by increasing the activity of pyruvate dehydrogenase kinases 1 and 4. Together with suppression of the catalytic subunit of pyruvate dehydrogenase phosphatase 1 this leads to increased phosphorylation of the E1α regulatory subunit of the pyruvate dehydrogenase complex, which in turn inhibits further oxidation of pyruvate in the mitochondria-instead, pyruvate is reduced to lactate. Suppression of FOXK1 and FOXK2 induce the opposite phenotype. Both in vitro and in vivo experiments, including studies of primary human cells, show how FOXK1 and/or FOXK2 are likely to act as important regulators that reprogram cellular metabolism to induce aerobic glycolysis.

PKM2 coordinates glycolysis with mitochondrial fusion and oxidative phosphorylation.

Abstract: A change in the metabolic flux of glucose from mitochondrial oxidative phosphorylation (OXPHOS) to aerobic glycolysis is regarded as one hallmark of cancer. However, the mechanisms underlying the metabolic switch between aerobic glycolysis and OXPHOS are unclear. Here we show that the M2 isoform of pyruvate kinase (PKM2), one of the rate-limiting enzymes in glycolysis, interacts with mitofusin 2 (MFN2), a key regulator of mitochondrial fusion, to promote mitochondrial fusion and OXPHOS, and attenuate glycolysis. mTOR increases the PKM2:MFN2 interaction by phosphorylating MFN2 and thereby modulates the effect of PKM2:MFN2 on glycolysis, mitochondrial fusion and OXPHOS. Thus, an mTOR-MFN2-PKM2 signaling axis couples glycolysis and OXPHOS to modulate cancer cell growth.

Pyruvate kinase M2 is requisite for Th1 and Th17 differentiation.

Abstract: Th1 and Th17 are important in the pathogenesis of autoimmune diseases and they depend on glycolysis as a source of energy. T cell antigen receptor signaling phosphorylates a serine/threonine kinase, calcium/calmodulin-dependent protein kinase IV (CaMK4), and promotes glycolysis. Based on these findings we hypothesized that CaMK4 promotes glycolysis. Camk4-deficient CD4+ T cells and cells treated with a CaMK4 inhibitor had less glycolysis compared with their counterparts. Pull-down of CaMK4 and mass spectrometry identified pyruvate kinase muscle isozyme (PKM), the final rate-limiting enzyme in glycolysis, as a binding partner. Coimmunoprecipitation and Western blotting showed that CaMK4 interacts directly with PKM2. Camk4-deficient CD4+ T cells displayed decreased pyruvate kinase activity. Silencing or pharmacological inhibition of PKM2 reduced glycolysis and in vitro differentiation to Th1 and Th17 cells, while PKM2 overexpression restored Th17 cell differentiation. Treatment with a PKM2 inhibitor ameliorated experimental autoimmune encephalomyelitis and CD4+ T cells treated with PKM2 inhibitor or Pkm2-shRNA caused limited disease activity in an adoptive cell transfer model of experimental autoimmune encephalomyelitis. Our data demonstrate that CaMK4 binds to PKM2 and promotes its activity, which is requisite for Th1 and Th17 differentiation in vitro and in vivo. PKM2 represents a therapeutic target for T cell-dependent autoimmune diseases.

An Overview of Structure, Function and Regulation of Pyruvate Kinases

Abstract: In the last step of glycolysis Pyruvate kinase catalyzes the irreversible conversion of ADP and phosphoenolpyruvate to ATP and pyruvic acid, both crucial for cellular metabolism. Thus pyruvate kinase plays a key role in controlling the metabolic flux and ATP production. The hallmark of the activity of different pyruvate kinases is their tight modulation by a variety of mechanisms including the use of a large number of physiological allosteric effectors in addition to their homotropic regulation by phosphoenolpyruvate. Binding of effectors signals precise and orchestrated movements in selected areas of the protein structure that alter the catalytic action of these evolutionarily conserved enzymes with remarkably conserved architecture and sequences. While the diverse nature of the allosteric effectors has been discussed in the literature, the structural basis of their regulatory effects is still not well understood because of the lack of data representing conformations in various activation states. Results of recent studies on pyruvate kinases of different families suggest that members of evolutionarily related families follow somewhat conserved allosteric strategies but evolutionarily distant members adopt different strategies. Here we review the structure and allosteric properties of pyruvate kinases of different families for which structural data are available.

Inhibition of mitochondrial pyruvate dehydrogenase kinase: a proposed mechanism by which melatonin causes cancer cells to overcome cytosolic glycolysis, reduce tumor biomass and reverse insensitivity to chemotherapy

Abstract: This review presents a hypothesis to explain the role of melatonin in regulating glucose metabolism in cancer cells. Many cancer cells use cytosolic glycolysis (the Warburg effect) to produce energy (ATP). Under these conditions, glucose is primarily converted to lactate which is released into the blood in large quantities. The Warburg effect gives cancer cells advantages in terms of enhanced macromolecule synthesis required for accelerated cellular proliferation, reduced cellular apoptosis which enhances tumor biomass and a greater likelihood of metastasis. Based on available data, high circulating melatonin levels at night serve as a signal for breast cancer cells to switch from cytosolic glycolysis to mitochondrial glucose oxidation and oxidative phosphorylation for ATP production. In this situation, melatonin promotes the synthesis of acetyl-CoA from pyruvate; we speculate that melatonin does this by inhibiting the mitochondrial enzyme pyruvate dehydrogenase kinase (PDK) which normally inhibits pyruvate dehydrogenase complex (PDC), the enzyme that controls the pyruvate to acetyl-CoA conversion. Acetyl-CoA has several important functions in the mitochondria; it feeds into the citric acid cycle which improves oxidative phosphorylation and, additionally, it is a necessary co-factor for the rate limiting enzyme, arylalkylamine N-acetyltransferase, in mitochondrial melatonin synthesis. When breast cancer cells are using cytosolic glycolysis (during the day) they are of the cancer phenotype; at night when they are using mitochondria to produce ATP via oxidative phosphorylation, they have a normal cell phenotype. If this day:night difference in tumor cell metabolism is common in other cancers, it indicates that these tumor cells are only cancerous part of the time. We also speculate that high nighttime melatonin levels also reverse the insensitivity of tumors to chemotherapy.

PKM2 is the target of proanthocyanidin B2 during the inhibition of hepatocellular carcinoma

Abstract: The treatment for advanced primary hepatocellular carcinoma (HCC) is sorafenib (SORA), while HCC has become increasingly drug resistant with enhanced aerobic glycolysis. The present study aimed to examine the chemotherapeutic effects of a flavonoid proanthocyanidin B2 (PB2) on HCC. Five kinds of HCC cell lines and LO2 were used to test the effect of PB2 on aerobic glycolysis. The proliferation, cell cycle, apoptosis and a xenograft mouse model were analyzed. Lentivirus overexpressed pyruvate kinase M2 (PKM2) or sh-PKM2 was used to verify the target of PB2. The detailed mechanism was investigated by immunofluorescence, co-immunoprecipitation, and western blotting. PB2 inhibited the proliferation, induced cell cycle arrest, and triggered apoptosis of HCC cells in vivo and in vitro. PB2 also suppressed glucose uptake and lactate levels via the direct inhibition of the key glycolytic enzyme, PKM2. In addition, PKM2 inhibited the nuclear translocation of PKM2 and co-localization of PKM2/HIF-1α in the nucleus, leading to the inhibition of aerobic glycolysis. Co-treatment with PB2 was also effective in enhancing the chemosensitivity of SORA. PB2 inhibited the expression and nuclear translocation of PKM2, therefore disrupting the interaction between PKM2/HSP90/HIF-1α, to suppress aerobic glycolysis and proliferation, and trigger apoptosis in HCC via HIF-1α-mediated transcription suppression.

Mitochondrial Oxidative Phosphorylation defect in the Heart of Subjects with Coronary Artery Disease.

Abstract: Coronary artery disease (CAD) is a leading cause of death worldwide and frequently associated with mitochondrial dysfunction. Detailed understanding of abnormalities in mitochondrial function that occur in patients with CAD is lacking. We evaluated mitochondrial damage, energy production, and mitochondrial complex activity in human non-CAD and CAD hearts. Fresh and frozen human heart tissue was used. Cell lysate or mitochondria were isolated using standard techniques. Mitochondrial DNA (mtDNA), NAD + and ATP levels, and mitochondrial oxidative phosphorylation capacity were evaluated. Proteins critical to the regulation of mitochondrial metabolism and function were also evaluated in tissue lysates. PCR analysis revealed an increase in mtDNA lesions and the frequency of mitochondrial common deletion, both established markers for impaired mitochondrial integrity in CAD compared to non-CAD patient samples. NAD+ and ATP levels were significantly decreased in CAD subjects compared to Non-CAD (NAD+ fold change: non-CAD 1.00 ± 0.17 vs. CAD 0.32 ± 0.12* and ATP fold change: non-CAD 1.00 ± 0.294 vs. CAD 0.01 ± 0.001*; N = 15, P < 0.005). We observed decreased respiration control index in CAD tissue and decreased activity of complexes I, II, and III. Expression of ETC complex subunits and respirasome formation were increased; however, elevations in the de-active form of complex I were observed in CAD. We observed a corresponding increase in glycolytic flux, indicated by a rise in pyruvate kinase and lactate dehydrogenase activity, indicating a compensatory increase in glycolysis for cellular energetics. Together, these results indicate a shift in mitochondrial metabolism from oxidative phosphorylation to glycolysis in human hearts subjects with CAD.

Knockdown of Pyruvate Kinase M Inhibits Cell Growth and Migration by Reducing NF-kB Activity in Triple-Negative Breast Cancer Cells.

Abstract: Altered genetic features in cancer cells lead to a high rate of aerobic glycolysis and metabolic reprogramming that is essential for increased cancer cell viability and rapid proliferation. Pyruvate kinase muscle (PKM) is a rate-limiting enzyme in the final step of glycolysis. Herein, we report that PKM is a potential therapeutic target in triple-negative breast cancer (TNBC) cells. We found that PKM1 or PKM2 is highly expressed in TNBC tissues or cells. Knockdown of PKM significantly suppressed cell proliferation and migration, and strongly reduced S phase and induced G2 phase cell cycle arrest by reducing phosphorylation of the CDC2 protein in TNBC cells. Additionally, knockdown of PKM significantly suppressed NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity by reducing the phosphorylation of p65 at serine 536, and also decreased the expression of NF-kB target genes. Taken together, PKM is a potential target that may have therapeutic implications for TNBC cells.

Prevalence and management of iron overload in pyruvate kinase deficiency: report from the Pyruvate Kinase Deficiency Natural History Study

Abstract: Pyruvate kinase (PK) deficiency is the most common red cell glycolytic enzyme defect causing hereditary non-spherocytic hemolytic anemia. Current treatments are mainly supportive and include red cell transfusions and splenectomy.[1][1] Regular red cell transfusions are known to result in iron

PKM2 promotes glucose metabolism through a let-7a-5p/Stat3/hnRNP-A1 regulatory feedback loop in breast cancer cells.

Abstract: Tumor cells metabolize more glucose to lactate in aerobic or hypoxic conditions than normal cells. Pyruvate kinase isoenzyme type M2 (PKM2) is crucial for tumor cell aerobic glycolysis. We established a role for let-7a-5p/Stat3/hnRNP-A1/PKM2 signaling in breast cancer cell glucose metabolism. PKM2 depletion via small interfering RNA (siRNA) inhibits cell proliferation and aerobic glycolysis in breast cancer cells. Signal transducer and activator of transcription 3 (Stat3) promotes upregulation of heterogeneous nuclear ribonucleoprotein (hnRNP)-A1 expression, hnRNP-A1 binding to pyruvate kinase isoenzyme (PKM) pre messenger RNA, and the subsequent formation of PKM2. This pathway is downregulated by the microRNA let-7a-5p, which functionally targets Stat3, whereas hnRNP-A1 blocks the biogenesis of let-7a-5p to counteract its ability to downregulate the Stat3/hnRNP-A1/PKM2 signaling pathway. The downregulation of Stat3/hnRNP-A1/PKM2 by let-7a-5p is verified using a breast cancer. These results suggest that let-7a-5p, Stat3, and hnRNP-A1 form a feedback loop, thereby regulating PKM2 expression to modulate glucose metabolism of breast cancer cells. These findings elucidate a new pathway mediating aerobic glycolysis in breast cancers and provide an attractive potential target for breast cancer therapeutic intervention.

Beta-elemene inhibits breast cancer metastasis through blocking pyruvate kinase M2 dimerization and nuclear translocation.

Abstract: Pyruvate kinase M2 (PKM2), playing a central role in regulating aerobic glycolysis, was considered as a promising target for cancer therapy. However, its role in cancer metastasis is rarely known. Here, we found a tight relationship between PKM2 and breast cancer metastasis, demonstrated by the findings that beta-elemene (β-elemene), an approved drug for complementary cancer therapy, exerted distinct anti-metastatic activity dependent on PKM2. The results indicated that β-elemene inhibited breast cancer cell migration, invasion in vitro as well as metastases in vivo. β-Elemene further inhibited the process of aerobic glycolysis and decreased the utilization of glucose and the production of pyruvate and lactate through suppressing pyruvate kinase activity by modulating the transformation of dimeric and tetrameric forms of PKM2. Further analysis revealed that β-elemene suppressed aerobic glycolysis by blocking PKM2 nuclear translocation and the expression of EGFR, GLUT1 and LDHA by influencing the expression of importin α5. Furthermore, the effect of β-elemene on migration, invasion, PKM2 transformation, and nuclear translocation could be reversed in part by fructose-1,6-bisphosphate (FBP) and L-cysteine. Taken together, tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis, and β-elemene inhibited breast cancer metastasis via blocking aerobic glycolysis mediated by dimeric PKM2 transformation and nuclear translocation, being a promising anti-metastatic agent from natural compounds.

Nitric oxide maintains endothelial redox homeostasis through PKM2 inhibition

Abstract: Decreased nitric oxide (NO) bioavailability and oxidative stress are hallmarks of endothelial dysfunction and cardiovascular diseases. Although numerous proteins are S-nitrosated, whether and how changes in protein S-nitrosation influence endothelial function under pathophysiological conditions remains unknown. We report that active endothelial NO synthase (eNOS) interacts with and S-nitrosates pyruvate kinase M2 (PKM2), which reduces PKM2 activity. PKM2 inhibition increases substrate flux through the pentose phosphate pathway to generate reducing equivalents (NADPH and GSH) and protect against oxidative stress. In mice, the Tyr656 to Phe mutation renders eNOS insensitive to inactivation by oxidative stress and prevents the decrease in PKM2 S-nitrosation and reducing equivalents, thereby delaying cardiovascular disease development. These findings highlight a novel mechanism linking NO bioavailability to antioxidant responses in endothelial cells through S-nitrosation and inhibition of PKM2.

Aerobic glycolysis fuels platelet activation: small-molecule modulators of platelet metabolism as anti-thrombotic agents.

Abstract: Platelets are critical to arterial thrombosis, which underlies myocardial infarction and stroke. Activated platelets, regardless of the nature of their stimulus, initiate energy-intensive processes that sustain thrombus, while adapting to potential adversities of hypoxia and nutrient deprivation within the densely packed thrombotic milieu. We report here that stimulated platelets switch their energy metabolism to aerobic glycolysis by modulating enzymes at key checkpoints in glucose metabolism. We found that aerobic glycolysis, in turn, accelerates flux through the pentose phosphate pathway and supports platelet activation. Hence, reversing metabolic adaptations of platelets could be an effective alternative to conventional anti-platelet approaches, which are crippled by remarkable redundancy in platelet agonists and ensuing signaling pathways. In support of this hypothesis, small-molecule modulators of pyruvate dehydrogenase, pyruvate kinase M2 and glucose-6-phosphate dehydrogenase, all of which impede aerobic glycolysis and/or the pentose phosphate pathway, restrained the agonist-induced platelet responses ex vivo. These drugs, which include the anti-neoplastic candidate, dichloroacetate, and the Food and Drug Administration-approved dehydroepiandrosterone, profoundly impaired thrombosis in mice, thereby exhibiting potential as anti-thrombotic agents.

Cysteine catabolism and the serine biosynthesis pathway support pyruvate production during pyruvate kinase knockdown in pancreatic cancer cells

Abstract: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with limited treatment options. Pyruvate kinase, especially the M2 isoform (PKM2), is highly expressed in PDAC cells, but its role in pancreatic cancer remains controversial. To investigate the role of pyruvate kinase in pancreatic cancer, we knocked down PKM2 individually as well as both PKM1 and PKM2 concurrently (PKM1/2) in cell lines derived from a KrasG12D/-; p53-/- pancreatic mouse model. We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine metabolic profiles of wildtype and PKM1/2 knockdown PDAC cells. We further used stable isotope-labeled metabolic precursors and LC-MS/MS to determine metabolic pathways upregulated in PKM1/2 knockdown cells. We then targeted metabolic pathways upregulated in PKM1/2 knockdown cells using CRISPR/Cas9 gene editing technology. PDAC cells are able to proliferate and continue to produce pyruvate despite PKM1/2 knockdown. The serine biosynthesis pathway partially contributed to pyruvate production during PKM1/2 knockdown: knockout of phosphoglycerate dehydrogenase in this pathway decreased pyruvate production from glucose. In addition, cysteine catabolism generated ~ 20% of intracellular pyruvate in PDAC cells. Other potential sources of pyruvate include the sialic acid pathway and catabolism of glutamine, serine, tryptophan, and threonine. However, these sources did not provide significant levels of pyruvate in PKM1/2 knockdown cells. PKM1/2 knockdown does not impact the proliferation of pancreatic cancer cells. The serine biosynthesis pathway supports conversion of glucose to pyruvate during pyruvate kinase knockdown. However, direct conversion of serine to pyruvate was not observed during PKM1/2 knockdown. Investigating several alternative sources of pyruvate identified cysteine catabolism for pyruvate production during PKM1/2 knockdown. Surprisingly, we find that a large percentage of intracellular pyruvate comes from cysteine. Our results highlight the ability of PDAC cells to adaptively rewire their metabolic pathways during knockdown of a key metabolic enzyme.

Pyruvate Homeostasis as a Determinant of Parasite Growth and Metabolic Plasticity in Toxoplasma gondii

Abstract: Toxoplasma gondii is a widespread intracellular pathogen infecting humans and a variety of animals. Previous studies have shown that Toxoplasma uses glucose and glutamine as the main carbon sources to support asexual reproduction, but neither nutrient is essential. Such metabolic flexibility may allow it to survive within diverse host cell types. Here, by focusing on the glycolytic enzyme pyruvate kinase (PYK) that converts phosphoenolpyruvate (PEP) into pyruvate, we found that Toxoplasma can also utilize lactate and alanine. We show that catabolism of all indicated carbon sources converges at pyruvate, and maintaining a constant pyruvate supply is critical to parasite growth. Toxoplasma expresses two PYKs: PYK1 in the cytosol and PYK2 in the apicoplast (a chloroplast relict). Genetic deletion of PYK2 did not noticeably affect parasite growth and virulence, which contrasts with the current model of carbon metabolism in the apicoplast. On the other hand, PYK1 was refractory to disruption. Conditional depletion of PYK1 resulted in global alteration of carbon metabolism, amylopectin accumulation, and reduced cellular ATP, leading to severe growth impairment. Notably, the attenuated growth of the PYK1-depleted mutant was partially rescued by lactate or alanine supplementation, and rescue by lactate required lactate dehydrogenase activity to convert it to pyruvate. Moreover, depletion of PYK1 in conjunction with PYK2 ablation led to accentuated loss of apicoplasts and complete growth arrest. Together, our results underline a critical role of pyruvate homeostasis in determining the metabolic flexibility and apicoplast maintenance, and they significantly extend our current understanding of carbon metabolism in T. gondii. IMPORTANCEToxoplasma gondii infects almost all warm-blooded animals, and metabolic flexibility is deemed critical for its successful parasitism in diverse hosts. Glucose and glutamine are the major carbon sources to support parasite growth. In this study, we found that Toxoplasma is also competent in utilizing lactate and alanine and, thus, exhibits exceptional metabolic versatility. Notably, all these nutrients need to be converted to pyruvate to fuel the lytic cycle, and achieving a continued pyruvate supply is vital to parasite survival and metabolic flexibility. Although pyruvate can be generated by two distinct pyruvate kinases, located in cytosol and apicoplast, respectively, the cytosolic enzyme is the main source of subcellular pyruvate, and cooperative usage of pyruvate among multiple organelles is critical for parasite growth and virulence. These findings expand our current understanding of carbon metabolism in Toxoplasma gondii and related parasites while providing a basis for designing novel antiparasitic interventions.