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Philip S. Low - One of the best experts on this subject based on the ideXlab platform.
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Mapping of Glycolytic Enzyme-binding sites on human erythrocyte band 3
The Biochemical journal, 2006Co-Authors: Haiyan Chu, Philip S. LowAbstract:Previous work has shown that GAPDH (glyceraldehyde-3-phosphate dehydrogenase), aldolase, PFK (phosphofructokinase), PK (pyruvate kinase) and LDH (lactate dehydrogenase) assemble into a GE (Glycolytic Enzyme) complex on the inner surface of the human erythrocyte membrane. In an effort to define the molecular architecture of this complex, we have undertaken to localize the binding sites of these Enzymes more accurately. We report that: (i) a major aldolase-binding site on the erythrocyte membrane is located within N-terminal residues 1–23 of band 3 and that both consensus sequences D6DYED10 and E19EYED23 are necessary to form a single Enzyme-binding site; (ii) GAPDH has two tandem binding sites on band 3, located in residues 1–11 and residues 12–23 respectively; (iii) a PFK-binding site resides between residues 12 and 23 of band 3; (iv) no GEs bind to the third consensus sequence (residues D902EYDE906) at the C-terminus of band 3; and (v) the LDH- and PK-binding sites on the erythrocyte membrane do not reside on band 3. Taken together, these results argue that band 3 provides a nucleation site for the GE complex on the human erythrocyte membrane and that other components near band 3 must also participate in organizing the Enzyme complex.
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Mapping of the Glycolytic Enzyme Binding Sites on Human Erythrocyte Membrane Band 3.
Blood, 2005Co-Authors: Haiyan Chu, Philip S. LowAbstract:Glycolytic Enzymes (GEs) including aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) are known to associate with the inner surface of the human erythrocyte membrane. Previous studies have suggested that the N-terminus of the cytoplasmic domain of band 3 (cdb3) constitutes the likely binding site for aldolase, GAPDH and PFK, however, the membrane docking sites for PK and LDH have not been elucidated. In this study, we demonstrate that PK and LDH exhibit no affinity for band 3, regardless of whether the association is measured by co-immunoprecipitation assay, binding competition studies, or catalytic inhibition analyses. We further find that the binding sites for GAPDH, aldolase and PFK on band 3 are distinct but partially overlapping, as evidenced by the fact that: 1) deletion of residues 1–11 of cdb3 eliminates the binding of aldolase, but not PFK or GAPDH, 2) fusion of thioredoxin (Trx) to the N-terminus of residues cdb3 blocks aldolase binding, but not the association of GAPDH or PFK, 3) deletion of sequences 1–50, 1–40, 1–31, or 1–23 of cdb3 blocks cdb3 association with all three GEs, whereas deletion of residues 12-23 only abrogates aldolase binding (while reducing the affinity of PFK and GAPDH), 4) the presence of both sequences, 6–DDYED-10 and 19-EEYED-23, are necessary for cdb3 association with aldolase, whereas the presence of either sequence alone (especially 19-EEYED-23) is sufficient to maintain association with GAPDH and PFK, 5) mutation of all of the acidic residues in the above two sequences to their corresponding amides (E→Q and D→N) results in loss of affinity for all GEs. Because i) kidney cdb3 (which lacks residues 1-65 of cdb3) shows no affinity for any of the GEs, ii) residues 1–55 of cdb3 show near normal affinity for aldolase, GAPDH and PFK, and iii) GAPDH, aldolase, and PFK all compete with each other for cdb3, we conclude that the binding sites for aldolase, GAPDH and PFK are all located within the first 23 residues of cdb3, with the docking site for aldolase likely residing somewhat more N-terminal than the binding sites for GAPDH and PFK. Finally, because each band 3 monomer contains three homologous sequences (6-DDYED-10, 19-EEYED-23 and 902-DEYDE-906) that are found in other proteins that bind GEs (e.g. actin, β-tubulin, troponin T), and since the first two of these sequences were shown to be required for cdb3 binding, the question naturally arose whether different GEs might also associate with residues 902-DEYDE-906 at the extreme C-terminus of band 3. Similar binding studies demonstrate that such an interaction does not occur. Thus, our data show that GAPDH, aldolase, and PFK all bind near the N-terminus of band 3 and that PK and LDH must dock somewhere else on the red cell membrane.
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Assembly and regulation of a Glycolytic Enzyme complex on the human erythrocyte membrane.
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: M. Estela Campanella, Haiyan Chu, Philip S. LowAbstract:To characterize the location of Glycolytic Enzymes (GEs) in intact human erythrocytes, freshly drawn blood was fixed and stained with Abs to GAPDH, aldolase, phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), carbonic anhydrase II, Hb, and band 3 (AE1). Confocal microscopy revealed that in cells where band 3 displays its expected membrane staining and Hb is evenly distributed across the cytoplasm, GEs are largely limited to the membrane. Biochemical studies confirmed that the membrane binding sites for GAPDH, aldolase, and PFK reside on band 3, but related analyses demonstrate that sites for PK and LDH do not. Four lines of evidence demonstrate that the GEs are at least partially assembled into multimeric complexes near the NH2 terminus of band 3. First, a mAb to residues 1–12 of band 3 displaces all of the above GEs from the membrane, including LDH and PK, which do not bind band 3. Second, tyrosine phosphorylation of the NH2 terminus of band 3 (Y8 and Y21) reversibly releases all of the GEs from the membrane, including LDH and PK. Third, deoxygenation of RBCs dislodges all GEs from the membrane, consistent with the established ability of deoxyHb but not oxyHb to bind the NH2 terminus of band 3. Fourth, a large increase in the accessibility of Enzyme epitopes is observed upon dissociation of GEs from the membrane. We conclude, therefore, that GEs are organized into complexes on the membrane whose assembly is regulated by oxygenation and phosphorylation.
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Characterization of Glycolytic Enzyme Complexes on Murine Erythrocyte Membranes.
Blood, 2004Co-Authors: Estela Campanella, Philip S. Low, William A. Anong, Cheryl A. Hillary, Nancy J WanderseeAbstract:Glycolytic Enzymes have been recently shown to exist as multi-Enzyme complexes in association with the cytoplasmic domain of band 3 at the inner surface of the human erythrocyte membrane. Because several of the Glycolytic Enzyme binding sites have been mapped to sequences near the NH2-terminus of band 3 (DDYED and EEYED) that are not conserved in mice (EEVLE and EELEN), the question naturally arose whether the existence of Glycolytic Enzyme complexes on erythrocyte membranes might be only a product of recent evolution. To test this hypothesis, fresh murine erythrocytes were fixed and stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH) and carbonic anhydrase II (CA II was used as a control, since it binds to a distant site near the COOH-terminus of band 3). Importantly, analysis of intact murine erythrocytes by confocal microscopy demonstrated that all of the above Enzymes are localized to the membrane in oxygenated cells. In contrast, upon deoxygenation of the intact cells, release of the Glycolytic Enzymes (but not CA II) from the erythrocyte membrane and their uniform redistribution throughout the cytoplasm is observed. Because deoxyhemoglobin has been shown in human erythrocytes to compete with Glycolytic Enzymes (but not with CA II) for a common binding site at the NH2-terminus of band 3, these data argue that murine band 3, despite its weak homology to human band 3, still constitutes an organization center for Glycolytic Enzymes on the erythrocyte membrane. To further test this hypothesis, erythrocytes from band 3 knockout mice were similarly examined by confocal microscopy. Not surprisingly, all of the Enzymes in all of the cells were evenly distributed throughout the cytoplasm, regardless of the oxygenation state of the cell. Further, immunoblot analyses demonstrated that Glycolytic Enzyme content of the band 3 knockout erythrocytes was measurably reduced compared to healthy mice, suggesting that the anion transporter may also contribute to Enzyme stabilization during the lifetime of the erythrocyte. Finally, to determine whether the integrity of other membrane structures might impact the assembly of Glycolytic Enzyme complexes on the erythrocyte membrane, α-spectrin deficient mice were also examined for their Enzyme distributions. Curiously, > 50% of the cells in any field exhibited Glycolytic Enzyme staining throughout the cytoplasm, with the remainder showing mainly membrane staining. Conceivably, the stabiity of Glycolytic Enzyme complexes on the membrane may also depend on the integrity of the membrane skeleton. Taken together, these data argue that Glycolytic Enzymes assemble in an oxygenation-dependent manner into complexes on murine erythrocyte membranes and that the stability of these complexes depends on the presence of band 3 and to a lesser extent α-spectrin. Supported by NIH grant GM24417.
Mauro Solapenna - One of the best experts on this subject based on the ideXlab platform.
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fructose 2 6 bisphosphate counteracts guanidinium chloride thermal and atp induced dissociation of skeletal muscle key Glycolytic Enzyme 6 phosphofructo 1 kinase a structural mechanism for pfk allosteric regulation
Archives of Biochemistry and Biophysics, 2007Co-Authors: Patricia Zancan, Fernanda V R Almeida, Joana Faberbarata, Joao M M Dellias, Mauro SolapennaAbstract:Abstract Rabbit muscle 6-phosphofructo-1-kinase (PFK) is the key Glycolytic Enzyme being regulated by diverse molecules and signals. This Enzyme may undergo a reversible dissociation from a fully active homotetramer to a quite inactive dimer. There are evidences that some positive and negative modulators of PFK, such as ADP and citrate, may interfere with the Enzyme oligomeric structure shifting the tetramer–dimer equilibrium towards opposite orientations, where the negative modulators favor the dissociation of tetramers into dimers and vice versa. PFK is allosterically inhibited by ATP at its physiological range of concentration, an effect counteracted by fructose-2,6-bisphosphate (F2,6BP). However, the structural molecular mechanism by which ATP and F2,6BP regulate PFK is hitherto demonstrated. The present paper aimed at demonstrating that either the ATP-induced inhibition of PFK and the reversion of this inhibition by F2,6BP occur through the same molecular mechanism, i.e., the displacement of the oligomeric equilibrium of the Enzyme. This conclusion is arrived assessing the effects of ATP and F2,6BP on PFK inactivation through two distinct ways to dissociate the Enzyme: (a) upon incubation at 50 °C, or (b) incubating the Enzyme with guanidinium hydrochloride (GdmCl). Our results reveal that temperature- and GdmCl-induced inactivation of PFK prove remarkably more effective in the presence 5 mM ATP than in the absence of additives. On the other hand, the presence of 100 nM F2,6BP attenuate the effects of both high-temperature exposition and GdmCl on PFK, even in the simultaneous presence of 5 mM ATP. These data support the hypothesis that ATP shifts the oligomeric equilibrium of PFK towards the smaller conformations, while F2,6BP acts in the opposite direction. This conclusion leads to important information about the molecular mechanism by which PFK is regulated by these modulators.
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clotrimazole inhibits and modulates heterologous association of the key Glycolytic Enzyme 6 phosphofructo 1 kinase
Biochemical Pharmacology, 2007Co-Authors: Patricia Zancan, Alicia O Rosas, Mariah C Marcondes, Monica M Marinhocarvalho, Mauro SolapennaAbstract:Clotrimazole is an antifungal azole derivative recently recognized as a calmodulin antagonist with promising anticancer effects. This property has been correlated with the ability of the drug to decrease the viability of tumor cells by inhibiting their Glycolytic flux and consequently decreasing the intracellular concentration of ATP. The effects of clotrimazole on cell glycolysis and ATP production are considered to be due to the detachment of the Glycolytic Enzymes from the cytoskeleton. Here, we show that clotrimazole directly inhibits the key Glycolytic Enzyme 6-phosphofructo-1-kinase (PFK). This property is independent of the anti-calmodulin activity of the drug, since it is not mimicked by the classical calmodulin antagonist compound 48/80. However, the clotrimazole-inhibited Enzyme can be activated by calmodulin, even though calmodulin has no effect on PFK activity in the absence of the drug. Clotrimazole alone induces the dimerization of PFK reducing the population of tetramers, which is not observed when calmodulin is also present. Since PFK dimers are less active than PFK tetramers, this can explain the inhibitory effect of clotrimazole on the Enzyme. Additionally, clotrimazole positively modulates the association of PFK with erythrocyte membranes. Altogether, our data support a hitherto unrecognized action of clotrimazole as a negative modulator of Glycolytic flux through direct inhibition of the key Enzyme PFK.
Patricia Zancan - One of the best experts on this subject based on the ideXlab platform.
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fructose 2 6 bisphosphate counteracts guanidinium chloride thermal and atp induced dissociation of skeletal muscle key Glycolytic Enzyme 6 phosphofructo 1 kinase a structural mechanism for pfk allosteric regulation
Archives of Biochemistry and Biophysics, 2007Co-Authors: Patricia Zancan, Fernanda V R Almeida, Joana Faberbarata, Joao M M Dellias, Mauro SolapennaAbstract:Abstract Rabbit muscle 6-phosphofructo-1-kinase (PFK) is the key Glycolytic Enzyme being regulated by diverse molecules and signals. This Enzyme may undergo a reversible dissociation from a fully active homotetramer to a quite inactive dimer. There are evidences that some positive and negative modulators of PFK, such as ADP and citrate, may interfere with the Enzyme oligomeric structure shifting the tetramer–dimer equilibrium towards opposite orientations, where the negative modulators favor the dissociation of tetramers into dimers and vice versa. PFK is allosterically inhibited by ATP at its physiological range of concentration, an effect counteracted by fructose-2,6-bisphosphate (F2,6BP). However, the structural molecular mechanism by which ATP and F2,6BP regulate PFK is hitherto demonstrated. The present paper aimed at demonstrating that either the ATP-induced inhibition of PFK and the reversion of this inhibition by F2,6BP occur through the same molecular mechanism, i.e., the displacement of the oligomeric equilibrium of the Enzyme. This conclusion is arrived assessing the effects of ATP and F2,6BP on PFK inactivation through two distinct ways to dissociate the Enzyme: (a) upon incubation at 50 °C, or (b) incubating the Enzyme with guanidinium hydrochloride (GdmCl). Our results reveal that temperature- and GdmCl-induced inactivation of PFK prove remarkably more effective in the presence 5 mM ATP than in the absence of additives. On the other hand, the presence of 100 nM F2,6BP attenuate the effects of both high-temperature exposition and GdmCl on PFK, even in the simultaneous presence of 5 mM ATP. These data support the hypothesis that ATP shifts the oligomeric equilibrium of PFK towards the smaller conformations, while F2,6BP acts in the opposite direction. This conclusion leads to important information about the molecular mechanism by which PFK is regulated by these modulators.
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clotrimazole inhibits and modulates heterologous association of the key Glycolytic Enzyme 6 phosphofructo 1 kinase
Biochemical Pharmacology, 2007Co-Authors: Patricia Zancan, Alicia O Rosas, Mariah C Marcondes, Monica M Marinhocarvalho, Mauro SolapennaAbstract:Clotrimazole is an antifungal azole derivative recently recognized as a calmodulin antagonist with promising anticancer effects. This property has been correlated with the ability of the drug to decrease the viability of tumor cells by inhibiting their Glycolytic flux and consequently decreasing the intracellular concentration of ATP. The effects of clotrimazole on cell glycolysis and ATP production are considered to be due to the detachment of the Glycolytic Enzymes from the cytoskeleton. Here, we show that clotrimazole directly inhibits the key Glycolytic Enzyme 6-phosphofructo-1-kinase (PFK). This property is independent of the anti-calmodulin activity of the drug, since it is not mimicked by the classical calmodulin antagonist compound 48/80. However, the clotrimazole-inhibited Enzyme can be activated by calmodulin, even though calmodulin has no effect on PFK activity in the absence of the drug. Clotrimazole alone induces the dimerization of PFK reducing the population of tetramers, which is not observed when calmodulin is also present. Since PFK dimers are less active than PFK tetramers, this can explain the inhibitory effect of clotrimazole on the Enzyme. Additionally, clotrimazole positively modulates the association of PFK with erythrocyte membranes. Altogether, our data support a hitherto unrecognized action of clotrimazole as a negative modulator of Glycolytic flux through direct inhibition of the key Enzyme PFK.
Haiyan Chu - One of the best experts on this subject based on the ideXlab platform.
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Mapping of Glycolytic Enzyme-binding sites on human erythrocyte band 3
The Biochemical journal, 2006Co-Authors: Haiyan Chu, Philip S. LowAbstract:Previous work has shown that GAPDH (glyceraldehyde-3-phosphate dehydrogenase), aldolase, PFK (phosphofructokinase), PK (pyruvate kinase) and LDH (lactate dehydrogenase) assemble into a GE (Glycolytic Enzyme) complex on the inner surface of the human erythrocyte membrane. In an effort to define the molecular architecture of this complex, we have undertaken to localize the binding sites of these Enzymes more accurately. We report that: (i) a major aldolase-binding site on the erythrocyte membrane is located within N-terminal residues 1–23 of band 3 and that both consensus sequences D6DYED10 and E19EYED23 are necessary to form a single Enzyme-binding site; (ii) GAPDH has two tandem binding sites on band 3, located in residues 1–11 and residues 12–23 respectively; (iii) a PFK-binding site resides between residues 12 and 23 of band 3; (iv) no GEs bind to the third consensus sequence (residues D902EYDE906) at the C-terminus of band 3; and (v) the LDH- and PK-binding sites on the erythrocyte membrane do not reside on band 3. Taken together, these results argue that band 3 provides a nucleation site for the GE complex on the human erythrocyte membrane and that other components near band 3 must also participate in organizing the Enzyme complex.
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Mapping of the Glycolytic Enzyme Binding Sites on Human Erythrocyte Membrane Band 3.
Blood, 2005Co-Authors: Haiyan Chu, Philip S. LowAbstract:Glycolytic Enzymes (GEs) including aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) are known to associate with the inner surface of the human erythrocyte membrane. Previous studies have suggested that the N-terminus of the cytoplasmic domain of band 3 (cdb3) constitutes the likely binding site for aldolase, GAPDH and PFK, however, the membrane docking sites for PK and LDH have not been elucidated. In this study, we demonstrate that PK and LDH exhibit no affinity for band 3, regardless of whether the association is measured by co-immunoprecipitation assay, binding competition studies, or catalytic inhibition analyses. We further find that the binding sites for GAPDH, aldolase and PFK on band 3 are distinct but partially overlapping, as evidenced by the fact that: 1) deletion of residues 1–11 of cdb3 eliminates the binding of aldolase, but not PFK or GAPDH, 2) fusion of thioredoxin (Trx) to the N-terminus of residues cdb3 blocks aldolase binding, but not the association of GAPDH or PFK, 3) deletion of sequences 1–50, 1–40, 1–31, or 1–23 of cdb3 blocks cdb3 association with all three GEs, whereas deletion of residues 12-23 only abrogates aldolase binding (while reducing the affinity of PFK and GAPDH), 4) the presence of both sequences, 6–DDYED-10 and 19-EEYED-23, are necessary for cdb3 association with aldolase, whereas the presence of either sequence alone (especially 19-EEYED-23) is sufficient to maintain association with GAPDH and PFK, 5) mutation of all of the acidic residues in the above two sequences to their corresponding amides (E→Q and D→N) results in loss of affinity for all GEs. Because i) kidney cdb3 (which lacks residues 1-65 of cdb3) shows no affinity for any of the GEs, ii) residues 1–55 of cdb3 show near normal affinity for aldolase, GAPDH and PFK, and iii) GAPDH, aldolase, and PFK all compete with each other for cdb3, we conclude that the binding sites for aldolase, GAPDH and PFK are all located within the first 23 residues of cdb3, with the docking site for aldolase likely residing somewhat more N-terminal than the binding sites for GAPDH and PFK. Finally, because each band 3 monomer contains three homologous sequences (6-DDYED-10, 19-EEYED-23 and 902-DEYDE-906) that are found in other proteins that bind GEs (e.g. actin, β-tubulin, troponin T), and since the first two of these sequences were shown to be required for cdb3 binding, the question naturally arose whether different GEs might also associate with residues 902-DEYDE-906 at the extreme C-terminus of band 3. Similar binding studies demonstrate that such an interaction does not occur. Thus, our data show that GAPDH, aldolase, and PFK all bind near the N-terminus of band 3 and that PK and LDH must dock somewhere else on the red cell membrane.
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Assembly and regulation of a Glycolytic Enzyme complex on the human erythrocyte membrane.
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: M. Estela Campanella, Haiyan Chu, Philip S. LowAbstract:To characterize the location of Glycolytic Enzymes (GEs) in intact human erythrocytes, freshly drawn blood was fixed and stained with Abs to GAPDH, aldolase, phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), carbonic anhydrase II, Hb, and band 3 (AE1). Confocal microscopy revealed that in cells where band 3 displays its expected membrane staining and Hb is evenly distributed across the cytoplasm, GEs are largely limited to the membrane. Biochemical studies confirmed that the membrane binding sites for GAPDH, aldolase, and PFK reside on band 3, but related analyses demonstrate that sites for PK and LDH do not. Four lines of evidence demonstrate that the GEs are at least partially assembled into multimeric complexes near the NH2 terminus of band 3. First, a mAb to residues 1–12 of band 3 displaces all of the above GEs from the membrane, including LDH and PK, which do not bind band 3. Second, tyrosine phosphorylation of the NH2 terminus of band 3 (Y8 and Y21) reversibly releases all of the GEs from the membrane, including LDH and PK. Third, deoxygenation of RBCs dislodges all GEs from the membrane, consistent with the established ability of deoxyHb but not oxyHb to bind the NH2 terminus of band 3. Fourth, a large increase in the accessibility of Enzyme epitopes is observed upon dissociation of GEs from the membrane. We conclude, therefore, that GEs are organized into complexes on the membrane whose assembly is regulated by oxygenation and phosphorylation.
Rameshwar N K Bamezai - One of the best experts on this subject based on the ideXlab platform.
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insulin enhances metabolic capacities of cancer cells by dual regulation of Glycolytic Enzyme pyruvate kinase m2
Molecular Cancer, 2013Co-Authors: Mohd Askandar Iqbal, Farid Ahmad Siddiqui, Vibhor Gupta, Shilpi Chattopadhyay, Prakasam Gopinath, Bhupender Kumar, Siddharth Manvati, Noor Chaman, Rameshwar N K BamezaiAbstract:Background Insulin is tightly associated with cancer progression; however, mechanistic insights into such observations are poorly understood. Recent studies show that metabolic transformation is critical to cancer cell proliferation. Here, we attempt to understand the role of insulin in promotion of cancer metabolism. To this end, the role of insulin in regulating Glycolytic Enzyme pyruvate kinase M2 (PKM2) was examined.
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insulin enhances metabolic capacities of cancer cells by dual regulation of Glycolytic Enzyme pyruvate kinase m2
Molecular Cancer, 2013Co-Authors: Mohd Askandar Iqbal, Farid Ahmad Siddiqui, Vibhor Gupta, Shilpi Chattopadhyay, Prakasam Gopinath, Bhupender Kumar, Siddharth Manvati, Noor Chaman, Rameshwar N K BamezaiAbstract:Insulin is tightly associated with cancer progression; however, mechanistic insights into such observations are poorly understood. Recent studies show that metabolic transformation is critical to cancer cell proliferation. Here, we attempt to understand the role of insulin in promotion of cancer metabolism. To this end, the role of insulin in regulating Glycolytic Enzyme pyruvate kinase M2 (PKM2) was examined. We observed that insulin up-regulated PKM2 expression, through PI3K/mTOR mediated HIF1α induction, but significantly reduced PKM2 activity independent of this pathway. Drop in PKM2 activity was attributed to subunit dissociation leading to formation of low activity PKM2 oligomers, as assessed by density gradient centrifugation. However, tyrosine 105 phosphorylation of PKM2, known for inhibiting PKM2 activity, remained unaffected on insulin treatment. Interestingly, insulin-induced ROS was found responsible for PKM2 activity reduction. The observed changes in PKM2 status led to augmented cancer metabolism. Insulin-induced PKM2 up-regulation resulted in enhanced aerobic glycolysis as confirmed by PKM2 knockdown studies. Further, PKM2 activity reduction led to characteristic pooling of Glycolytic intermediates and increased accumulation of NADPH; suggesting diversion of glucose flux towards macromolecular synthesis, necessary for cancer cell growth. The study identifies new PKM2-mediated effects of insulin on cancer metabolism, thus, advancing the understanding of insulin’s role in cancer.