Effect of phorbol myristate acetate and concanavalin A on the glycolytic enzymes of human peripheral lymphocytes

doi:10.1016/0167-4889(88)90215-7

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

Volume 970, Issue 1, 8 June 1988, Pages 1-6

https://doi.org/10.1016/0167-4889(88)90215-7 Get rights and content

Abstract

The effects of Concanavalin A and the tumor promoting agent, phorbol 12-myristate 13-acetate (PMA), on glycolytic enzymes in human peripheral lymphocytes have been studied. A combination of Concanavalin A plus PMA stimulates DNA and protein synthesis to a significantly greater extent than when each are added individually. PMA and concanavalin A together, but not individually, also increase the levels of the activity of the glycolytic enzymes in peripheral lymphocytes treated for 48 h. The increase in hexokinase activity induced by PMA plus concanavalin A appeared to be due to the expression of the isoenzyme form, hexokinase II. The results suggest that the expression of glycolytic enzymes in stimulated lymphocytes is a late event (perhaps associated with the S phase) which is regulated by a cellular signal system controlled by the combined action of PMA plus concanavalin A.

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Citation Excerpt :
In mitogen-stimulated murine lymphocytes, DNA synthesis (as measured by 3H-thymidine incorporation) parallels the rate of lactate production for the 2 days post-stimulation [18]. In a similar manner, mitogen-stimulated human peripheral lymphocytes showed that glycolytic enzyme activities, lactate production and DNA synthesis reached maximum levels in S phase of the cell cycle [37]. As well, anti-CD3+/anti-CD28+ stimulation of human peripheral T cells significantly induced lymphocytes to enter into S-phase and undergo cellular proliferation [38].
Activated T cells undergo metabolic reprogramming which promotes glycolytic flux and lactate production as well as elevated production of lipids, proteins, nucleic acids and other carbohydrates (i.e. induction of biomass) even in the presence of oxygen. Activated T cells show induced expression of, among other things, Glucose Transporter 1 and several glycolytic enzymes, including ADP-Dependent Glucokinase and the low affinity isoform Pyruvate Kinase-M2 (which promote glycolytic flux), as well Glutamine Transporters and Glycerol-3-phosphate Dehydrogenase 2 which make available glutamate and glycerol-3-phosphate as mitochondrial energy sources. Intracellular leucine concentrations critically regulate mammalian target of rapamycin (mTOR) signaling to promote Th1, Th2, and Th17 CD4⁺ T effector cell differentiation. In contrast, T regulatory (Treg) cells are generated when AMP-Activating Protein Kinase signaling is activated and mTOR activation is suppressed. Unlike effector CD4⁺ and CD8⁺ T cells, Tregs and memory T cells oxidize fatty acids for fuel. Effector and memory T cells perform different functions and thus show distinct metabolic profiles which are exquisitely controlled by cellular signaling. Upon activation, T cells express the insulin and leptin receptors on their surface and become sensitive to insulin signaling and nutrient availability and show changes in differentiation. Thus, metabolism and nutrient availability influence T cell activation and function.
The role of multiple enzyme activation in metabolic flux control
1998, Advances in Enzyme Regulation
Over recent years, the concept that flux through a metabolic pathway is controlled by a single rate-limiting enzyme has been challenged from a number of quarters. We have presented three lines of evidence that the control of pathway flux by activation of multiple several steps, including steps responsible for consumption of pathway products, is an important feature of physiological flux control in response to external stimmuli. Theoretical tools exist that can be used to analyze the distribution of flux control between the steps involved in signal transduction and enzyme activation.
Energy levels in resting and mitogen-stimulated human lymphocytes during treatment with FK506 or cyclosporin A in vitro
1997, Biochimica et Biophysica Acta - Bioenergetics
By employing microcalorimetry to assess overall metabolic activity in combination with other assays for specific metabolic events, we have investigated the influence of cyclosporin A and FK506 on the metabolic status of resting and mitogen-stimulated human peripheral lymphocytes. Both cyclosporin A and FK506 significantly reduced heat output from resting lymphocytes. This reduction could not be correlated with effects on DNA synthesis, lactate production, ATP levels or mitochondrial uptake of Rhodamine 123. These two drugs also potently reduced the increase in heat output seen during mitogen stimulation of lymphocytes. Both cyclosporin A and FK506 also prevented the increase in DNA synthesis, lactate production and ATP levels seen in response to mitogen stimulation. The increase in mitochondrial uptake of Rhodamine 123 during blastoid transformation was significantly reduced only by cyclosporin A. We ascribe the major part of the effects of these compounds to inhibition of the glycolytic pathway in both resting and mitogen-stimulated lymphocytes. These results indicate that the immunosuppressants cyclosporin A and FK506 exert other effects on lymphocytes than their well-established inhibitory action on calcineurin.© 1997 Elsevier Science B.V. All rights reserved.
Cell cycle-related changes in F-actin distribution are correlated with glycolytic activity
1995, Experimental Cell Research
XTH-2 cells, a cell line derived from tadpole heart endothelial cells, were blocked at the end of the G₁ phase of the cell cycle using desoxyguanosine (dG). Stress fibers are the dominant actin fibril structure in blocked cells. They disappear starting with the release of the dG block until the G₂ phase of the cell cycle. In addition, after the release peripheral lamellae and microspikes appear, indicating increased surface motility of the cells. Concomitantly with these changes, net lactic acid production is increased while oxygen consumption remains constant. Increase in the content of F-actin per cell takes place only in subconfluent cultures. Similar morphological changes (organization of F-actin) have been induced by phorbol myristate acetate treatment. These are also accompanied by an increase in lactic acid production and unaltered oxygen consumption. Therefore, the changes in energy metabolism are supposed to result from the rearrangement of the F-actin network: The emanating loose fibrillar pattern provides an increased surface for the association with glycolytic enzymes, which enhances enzyme activity and represents a more motile state. The increased energy demand of the more motile structures is supplied predominantly by ATP derived from glycolysis.
Expression of glycolytic isozymes in rat thymocytes during cell cycle progression
1994, BBA - Molecular Cell Research
The time courses of activities of aldolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase and pyruvate kinase were determined in stimulated rat thymocytes at 24 h intervals during a period of 72 h of culture. In parallel the mRNA levels of these enzymes were analysed by Northern blotting with specific probes. Both the enzyme activities and the corresponding mRNA levels reached their maxima 48 h after stimulation coinciding with the S-phase of the cell cycle. The isozyme types of aldolase and hexokinase in resting and in mitogen-stimulated rat thomocytes were identified by Northern blot hybridisation using isozyme-specific probes. In these cells the aldolase A is expressed, whereas type B and C could not be detected. The transcription of the aldolase A gene can be regulated by two different promoters. Depending on the alternative usage of the promoters the aldolase A-specific mRNA either contains the non-translated exons M1 or AH1. In rat thymocytes the promoter proximal to the exon AH1 is used while the expression of mRNA I, the type characteristic for muscle tissue, was not observed. In contrast to aldolase two isozyme types of hexokinase were detected. Hexokinase I as well as hexokinase II were present in thymocytes whereas hexokinase III was not detectable. A shift in the isosozyme pattern was not observed during the cell cycle progression.
Expression of glycolytic isoenzymes in activated human peripheral lymphocytes: Cell cycle analysis using flow cytometry
1991, Experimental Cell Research
Human peripheral lymphocytes activated with concanavalin A and phorbol myristate ester exhibit an increase in glycolysis on a time-course similar to that for DNA synthesis. Elevated glycolysis is accompanied by increased specific activities of the glycolytic enzymes. Increased enzyme activities are accounted for by the appearance of specific isoenzyme forms (muscle forms) normally expressed in rapidly growing tumor cells or in growth-stimulated cells. In the present study we analyzed the expression of the glycolytic isoenzymes during cell cycle progression of activated human lymphocytes using two-parameter (DNA and protein) flow cytometry. Time-course studies and analysis of subpopulations prepared by elutriation centrifugation showed that the inducible isoenzymes are expressed at low levels or not at all in G₀ cells. They are expressed first during the G₀ to G₁ transition or in early G₁. However, expression increases throughout G₁, reaching a maximum in S-phase. Thus, induction of glycolytic isoenzymes provides an excellent marker of T-cell activation and progression toward DNA synthesis.

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Regular paperEffect of phorbol myristate acetate and concanavalin A on the glycolytic enzymes of human peripheral lymphocytes

Abstract

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Regular paper
Effect of phorbol myristate acetate and concanavalin A on the glycolytic enzymes of human peripheral lymphocytes